Genes and uses for plant improvement

ABSTRACT

Transgenic seed for crops with improved traits are provided by trait-improving recombinant DNA in the nucleus of cells of the seed where plants grown from such transgenic seed exhibit one or more improved traits as compared to a control plant. Of particular interest are transgenic plants that have increased yield. The present invention also provides recombinant DNA molecules for expression of a protein, and recombinant DNA molecules for suppression of a protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 USC §119(e) of U.S. provisionalapplication Ser. No. 60/873,247, filed Dec. 6, 2006, herein incorporatedby reference.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Copy 1 and Copy 2) and a computerreadable form (CRF) of the sequence listing, all on CD-Rs, eachcontaining the file named 3126005US4.TXT, which is 67,123,200 bytes(measured in MS-WINDOWS) and was created on Mar. 7, 2016, areincorporated herein by reference in their entirety.

INCORPORATION OF COMPUTER PROGRAM LISTING

Two copies of Computer Program Listing (Copy 1 and Copy 2) containingfolders “hmmer-2.3.2” and “161pfamDir” are provided on separate CD-ROMsand are incorporated herein by reference in their entirety. Folderhmmer-2.3.2 contains the source code and other associated file forimplementing the HMMer software for Pfam analysis. Folder 161pfamDircontains 161 profile Hidden Markov Models. Both folders were created onthe disk on Mar. 2, 2016 having a total size of 15,534,080 bytes whenmeasured in MS-WINDOWS® operating system.

FIELD OF THE INVENTION

Disclosed herein are transgenic plant cells, plants and seeds comprisingrecombinant DNA and methods of making and using such plant cells, plantsand seeds

BACKGROUND OF THE INVENTION

Transgenic plants with enhanced traits such as improved yield,environmental stress tolerance, pest resistance, herbicide tolerance,modified seed compositions, and the like are desired by both farmers andconsumers. Although considerable efforts in plant breeding have providedsignificant gains in desired traits, the ability to introduce specificDNA into plant genomes provides further opportunities for generation ofplants with improved and/or unique traits. The ability to developtransgenic plants with improved traits depends in part on theidentification of useful recombinant DNA for production of transformedplants with improved properties, e.g. by actually selecting a transgenicplant from a screen for such improved property. An object of thisinvention is to provide transgenic plant cell nuclei, plant cells,plants and seeds by screening transgenic crop plants for one of moreenhanced agronomic traits where the nucleus in cells of the plant orseed has recombinant DNA that was identified as imparting an improvedtrait in a model plant, e.g. Arabidopsis thaliana. In some cases themodel plant may exhibit an improved trait that corresponds to anenhanced agronomic trait, e.g. cold stress tolerance, water deficitstress tolerance, low nitrogen stress tolerance and the like. In othercases the model plant may exhibit an improved trait that is a surrogateto an enhanced agronomic trait, e.g. salinity stress tolerance being asurrogate to drought tolerance or improvement in plant growth anddevelopment being a surrogate to enhanced yield. A further object of theinvention is to provide screening methods requiring routineexperimentation by which such transgenic plant cell nuclei, cells,plants and seeds can be identified by making a reasonable number oftransgenic events and engaging in screening identified in thisspecification and illustrated in the examples.

SUMMARY OF THE INVENTION

This invention provides plant cell nuclei with recombinant DNA thatimparts enhanced agronomic traits in transgenic plants having the nucleiin their cells. Recombinant DNA in this invention is provided in aconstruct comprising a promoter that is functional in plant cells andthat is operably linked to DNA that encodes a protein having at leastone amino acid domain in a sequence that exceeds the Pfam gatheringcutoff for amino acid sequence alignment with a protein domain familyidentified by a Pfam name in the group of Pfam domain names identifiedin Table 17. In more specific embodiments of the invention plant cellsare provided which express a protein having amino acid sequence with atleast 90% identity to a consensus amino acid sequence in the group ofconsensus amino acid sequences consisting of the consensus amino acidsequence constructed for SEQ ID NO: 198 and homologs thereof listed inTable 2 through the consensus amino acid sequence constructed for SEQ IDNO: 394 and homologs thereof listed in Table 2. Amino acid sequences ofhomologs are SEQ ID NO: 395 through 19,938. In even more specificembodiments of the invention the protein expressed in plant cells is aprotein selected from the group of proteins identified in Table 1 byannotation to a related protein in Genbank and alternatively identifiedin Table 16 by identification of protein domain family.

Other aspects of the invention are specifically directed to transgenicplant cells, and transgenic plants comprising a plurality of plant cellswith such nuclei, progeny transgenic seed, embryo and transgenic pollenfrom such plants. Such plant cell nuclei are selected from a populationof transgenic plants regenerated from plant cells with a nucleustransformed with recombinant DNA by screening the transgenic plants inthe population for an enhanced trait as compared to control plants thatdo not have the recombinant DNA in their nucleus, where the enhancedtrait is enhanced water use efficiency, enhanced cold tolerance,enhanced heat tolerance, enhanced shade tolerance, enhanced tolerance tosalt exposure, increased yield, enhanced nitrogen use efficiency,enhanced seed protein or enhanced seed oil. In some aspects of theinvention the recombinant DNA expresses a protein that imparts theenhanced trait; in other aspects of the invention the recombinant DNAexpresses RNA for suppressing the level of an endogenous protein. In yetanother aspect of the invention the nucleus of plant cells in plants,seeds, embryo and pollen further comprise DNA expressing a protein thatprovides tolerance from exposure to an herbicide applied at levels thatare lethal to a wild type plant cell. Such tolerance is especiallyuseful not only as an advantageous trait in such plants but is alsouseful in a selection step in the methods of the invention. In aspectsof the invention the agent of such herbicide is a glyphosate, dicamba,or glufosinate compound.

Yet other aspects of the invention provide nuclei in cells of transgenicplants which are homozygous for the recombinant DNA and transgenic seedof the invention from corn, soybean, cotton, canola, alfalfa, wheat orrice plants.

In other embodiments for practice of various aspects of the invention inArgentina the recombinant DNA in the nucleus is provided in plant cellsderived from corn lines that are and maintain resistance to a virus suchas the Mal de Rio Cuarto virus or a fungus such as the Puccinia sorghifungus or to both.

This invention also provides methods for manufacturing non-natural,transgenic seed that can be used to produce a crop of transgenic plantswith an enhanced trait resulting from expression of stably-integrated,recombinant DNA in the nucleus of the plant cells. In some aspects ofthe invention the recombinant DNA can express a protein having at leastone domain of amino acids in a sequence that exceeds the Pfam gatheringcutoff for amino acid sequence alignment with a protein domain familyidentified by a Pfam name in the group of Pfam names identified in Table17; in other aspects the recombinant DNA suppresses the level of such aprotein. More specifically the method comprises (a) screening apopulation of plants for an enhanced trait and recombinant DNA, whereindividual plants in the population can exhibit the trait at a levelless than, essentially the same as or greater than the level that thetrait is exhibited in control plants which do not express therecombinant DNA; (b) selecting from the population one or more plantsthat exhibit the trait at a level greater than the level that said traitis exhibited in control plants; (c) verifying that the recombinant DNAis stably integrated in said selected plants; (d) analyzing tissue of aselected plant to determine the production of a protein having thefunction of a protein encoded by nucleotides in a sequence of one of SEQID NO: 1-197; and (e) collecting seed from a selected plant. In oneaspect of the invention the plants in the population further compriseDNA expressing a protein that provides tolerance to exposure to anherbicide applied at levels that are lethal to wild type plant cells andwhere the selecting is effected by treating the population with theherbicide, e.g. a glyphosate, dicamba, or glufosinate compound. Inanother aspect of the invention the plants are selected by identifyingplants with the enhanced trait. The methods are especially useful formanufacturing corn, soybean, cotton, alfalfa, wheat or rice seedselected as having at least one of the enhanced traits described above.

Another aspect of the invention provides a method of producing hybridcorn seed comprising acquiring hybrid corn seed from a herbicidetolerant corn plant which also has a nucleus of this invention withstably-integrated, recombinant DNA The method further comprisesproducing corn plants from said hybrid corn seed, where a fraction ofthe plants produced from said hybrid corn seed is homozygous for saidrecombinant DNA, a fraction of the plants produced from said hybrid cornseed is hemizygous for said recombinant DNA, and a fraction of theplants produced from said hybrid corn seed has none of said recombinantDNA; selecting corn plants which are homozygous and hemizygous for saidrecombinant DNA by treating with an herbicide; collecting seed fromherbicide-treated-surviving corn plants and planting said seed toproduce further progeny corn plants; repeating the selecting andcollecting steps at least once to produce an inbred corn line; andcrossing the inbred corn line with a second corn line to produce hybridseed.

Another aspect of the invention provides a method of selecting a plantcomprising a nucleus of this invention in its plant cells by using animmunoreactive antibody to detect the presence of protein expressed byrecombinant DNA in seed or plant tissue. Another aspect of the inventionprovides anti-counterfeit milled seed having, as an indication oforigin, a nucleus of this invention with unique recombinant DNA.

Aspects of the invention relating to nucleus in plant cells havingrecombinant DNA for suppressing the expression of a protein areidentified in Table 1 and Table 16. More specific aspects of theinvention provide plant cells having recombinant DNA for suppressing theexpression of a protein having the function in a plant of the proteinwith amino acid sequence of SEQ ID NO: 200, 201, 205, 207, 211 and 394or the corresponding Pfam identified in Table 16, i.e. SNF5, LMBR1,TFIIS_M, TFIIS_C, Glyco_transf_8, respectively. Such suppression can beeffected by any of a number of ways known in the art, e.g. anti-sensesuppression, RNAi or mutation knockout and the like.

Another aspect of this invention relates to growing transgenic plantswith enhanced water use efficiency or enhanced nitrogen use efficiency.For instance, this invention provides methods of growing a corn, cottonor soybean crop without irrigation water comprising planting seed havingplant cells of the invention which are selected for enhanced water useefficiency. Alternatively methods comprise applying reduced irrigationwater, e.g. providing up to 300 millimeters of ground water during theproduction of a corn crop. This invention also provides methods ofgrowing a corn, cotton or soybean crop without added nitrogen fertilizercomprising planting seed having plant cells of the invention which areselected for enhanced nitrogen use efficiency. Alternatively methodscomprise applying reduced amount of nitrogen input as compared to theconventional input during the production of a corn crop.

The various aspects of this invention are especially useful fortransgenic plant cells in seeds and transgenic plants having any of theabove-described enhanced traits in crop plants such as corn (maize),soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa,barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.

The invention also provides recombinant DNA constructs comprising theDNA useful in the nuclei in plant cells for imparting enhanced traits inplants having those cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1N illustrates a consensus amino acid sequence of SEQ ID NO: 227and its homologs.

FIGS. 2, 3 and 4 illustrate plasmid maps.

DETAILED DESCRIPTION OF THE INVENTION

In the attached sequence listing:

SEQ ID NO: 1-197 are nucleotide sequences of the coding strand of DNAfor “genes” used in the recombinant DNA imparting an enhanced trait inplant cells, i.e. each represents a coding sequence for a protein;

SEQ ID NO: 198-394 are amino acid sequences of the cognate protein ofthe “genes” with nucleotide coding sequence 1-197;

SEQ ID NO: 395-19938 are amino acid sequences of homologous proteins;

SEQ ID NO: 19939 is a consensus amino acid sequence.

SEQ ID NO: 19940 is a nucleotide sequence of a plasmid base vectoruseful for corn transformation; and

SEQ ID NO: 19941 is a DNA sequence of a plasmid base vector useful forsoybean or canola transformation.

SEQ ID NO:19942 is a DNA sequence of a plasmid base vector useful forcotton transformation.

The nuclei of this invention are identified by screening transgenicplants for one or more traits including improved drought stresstolerance, improved heat stress tolerance, improved cold stresstolerance, improved high salinity stress tolerance, improved lownitrogen availability stress tolerance, improved shade stress tolerance,improved plant growth and development at the stages of seed imbibitionthrough early vegetative phase, and improved plant growth anddevelopment at the stages of leaf development, flower production andseed maturity.

“Gene” refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, orother DNA that encodes a peptide, polypeptide, protein, or RNA molecule,and regions flanking the coding sequences involved in the regulation ofexpression. In aspects of the invention where an improved trait isprovided by expression of a protein, “gene” refers at least to codingnucleotide sequence for a protein or a functional polypeptide fragmentof a protein that imparts the trait. In aspects of the invention wherean improved trait is provided by suppression of expression of anendogenous protein, “gene” refers to any part of the gene that can be atarget for suppression.

“Transgenic seed” means a plant seed whose nucleus has been altered bythe incorporation of recombinant DNA, e.g., by transformation asdescribed herein. The term “transgenic plant” is used to refer to theplant produced from an original transformation event, or progeny fromlater generations or crosses of a plant to a transformed plant, so longas the progeny contains a nucleus with the recombinant DNA in itsgenome.

“Recombinant DNA” means a polynucleotide having a genetically engineeredmodification introduced through combination of endogenous and/orexogenous elements in a transcription unit, manipulation viamutagenesis, restriction enzymes, and the like or simply by insertingmultiple copies of a native transcription unit. Recombinant DNA maycomprise DNA segments obtained from different sources, or DNA segmentsobtained from the same source, but which have been manipulated to joinDNA segments which do not naturally exist in the joined form. Arecombinant polynucleotide may exist outside of the cell, for example asa PCR fragment, or integrated into a genome, such as a plant genome.

“Trait” means a physiological, morphological, biochemical, or physicalcharacteristic of a plant or particular plant material or cell. In someinstances, this characteristic is visible to the human eye, such as seedor plant size, or can be measured by biochemical techniques, such asdetecting the protein, starch, or oil content of seed or leaves, or byobservation of a metabolic or physiological process, e.g., by measuringuptake of carbon dioxide, or by the observation of the expression levelof a gene or genes, e.g., by employing Northern analysis, RT-PCR,microarray gene expression assays, or reporter gene expression systems,or by agricultural observations such as stress tolerance, yield, orpathogen tolerance.

A “control plant” is a plant without trait-improving recombinant DNA inits nucleus. A control plant is used to measure and compare traitimprovement in a transgenic plant with such trait-improving recombinantDNA. A suitable control plant may be a non-transgenic plant of theparental line used to generate a transgenic plant herein. Alternatively,a control plant may be a transgenic plant that comprises an empty vectoror marker gene, but does not contain the recombinant DNA that producesthe trait improvement. A control plant may also be a negative segregantprogeny of hemizygous transgenic plant. In certain demonstrations oftrait improvement, the use of a limited number of control plants cancause a wide variation in the control dataset. To minimize the effect ofthe variation within the control dataset, a “reference” is used. As useherein a “reference” is a trimmed mean of all data from both transgenicand control plants grown under the same conditions and at the samedevelopmental stage. The trimmed mean is calculated by eliminating aspecific percentage, e.g., 20%, of the smallest and largest observationfrom the data set and then calculating the average of the remainingobservation.

“Trait enhancement” means a detectable and desirable difference in acharacteristic in a transgenic plant relative to a control plant or areference. In some cases, the trait enhancement can be measuredquantitatively. For example, the trait improvement can entail at least a2% desirable difference in an observed trait, at least a 5% desirabledifference, at least about a 10% desirable difference, at least about a20% desirable difference, at least about a 30% desirable difference, atleast about a 50% desirable difference, at least about a 70% desirabledifference, or at least about a 100% difference, or an even greaterdesirable difference. In other cases, the trait enhancement is onlymeasured qualitatively. It is known that there can be a naturalvariation in a trait. Therefore, the trait enhancement observed entailsa change of the normal distribution of the trait in the transgenic plantcompared with the trait distribution observed in a control plant or areference, which is evaluated by statistical methods provided herein.Trait enhancement includes, but is not limited to, yield increase,including increased yield under non-stress conditions and increasedyield under environmental stress conditions. Stress conditions mayinclude, for example, drought, shade, fungal disease, viral disease,bacterial disease, insect infestation, nematode infestation, coldtemperature exposure, heat exposure, osmotic stress, reduced nitrogennutrient availability, reduced phosphorus nutrient availability and highplant density.

Many agronomic traits can affect “yield”, including without limitation,plant height, pod number, pod position on the plant, number ofinternodes, incidence of pod shatter, grain size, efficiency ofnodulation and nitrogen fixation, efficiency of nutrient assimilation,resistance to biotic and abiotic stress, carbon assimilation, plantarchitecture, resistance to lodging, percent seed germination, seedlingvigor, and juvenile traits. Other traits that can affect yield include,efficiency of germination (including germination in stressedconditions), growth rate (including growth rate in stressed conditions),ear number, seed number per ear, seed size, composition of seed (starch,oil, protein) and characteristics of seed fill. Also of interest is thegeneration of transgenic plants that demonstrate desirable phenotypicproperties that may or may not confer an increase in overall plantyield. Such properties include enhanced plant morphology, plantphysiology or improved components of the mature seed harvested from thetransgenic plant.

“Yield-limiting environment” means the condition under which a plantwould have the limitation on yield including environmental stressconditions.

“Stress condition” means a condition unfavorable for a plant, whichadversely affect plant metabolism, growth and/or development. A plantunder the stress condition typically shows reduced germination rate,retarded growth and development, reduced photosynthesis rate, andeventually leading to reduction in yield. Specifically, “water deficitstress” used herein preferably refers to the sub-optimal conditions forwater and humidity needed for normal growth of natural plants. Relativewater content (RWC) can be used as a physiological measure of plantwater deficit. It measures the effect of osmotic adjustment in plantwater status, when a plant is under stress conditions. Conditions thatmay result in water deficit stress include heat, drought, high salinityand PEG induced osmotic stress.

“Cold stress” means the exposure of a plant to a temperatures below (twoor more degrees Celsius below) those normal for a particular species orparticular strain of plant.

“Nitrogen nutrient” means any one or any mix of the nitrate saltscommonly used as plant nitrogen fertilizer, including, but not limitedto, potassium nitrate, calcium nitrate, sodium nitrate, ammoniumnitrate. The term ammonium as used herein means any one or any mix ofthe ammonium salts commonly used as plant nitrogen fertilizer, e.g.,ammonium nitrate, ammonium chloride, ammonium sulfate, etc.

“Low nitrogen availability stress” means a plant growth condition thatdoes not contain sufficient nitrogen nutrient to maintain a healthyplant growth and/or for a plant to reach its typical yield under asufficient nitrogen growth condition. For example, a low nitrogencondition can refers to a growth condition with 50% or less of theconventional nitrogen inputs. “Sufficient nitrogen growth condition”means a growth condition where the soil or growth medium contains orreceives optimal amounts of nitrogen nutrient to sustain a healthy plantgrowth and/or for a plant to reach its typical yield for a particularplant species or a particular strain. One skilled in the art wouldrecognize what constitute such soil, media and fertilizer inputs formost plant species.

“Shade stress” means a growth condition that has limited lightavailability that triggers the shade avoidance response in plant. Plantsare subject to shade stress when localized at lower part of the canopy,or in close proximity of neighboring vegetation. Shade stress may becomeexacerbated when the planting density exceeds the average prevailingdensity for a particular plant species. The average prevailing densitiesper acre of a few examples of crop plants in the USA in the year 2000were: wheat 1,000,000-1,500,000; rice 650,000-900,000; soybean150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000 andcotton 28,000-55,000 plants per acre (Cheikh, e.g., (2003) U.S. PatentApplication No. 20030101479).

“Increased yield” of a transgenic plant of the present invention isevidenced and measured in a number of ways, including test weight, seednumber per plant, seed weight, seed number per unit area (i.e., seeds,or weight of seeds, per acre), bushels per acre, tons per acre, tons peracre, kilo per hectare. For example, maize yield can be measured asproduction of shelled corn kernels per unit of production area, e.g., inbushels per acre or metric tons per hectare, often reported on amoisture adjusted basis, e.g., at 15.5% moisture. Increased yield canresult from improved utilization of key biochemical compounds, such asnitrogen, phosphorous and carbohydrate, or from improved tolerance toenvironmental stresses, such as cold, heat, drought, salt, and attack bypests or pathogens. Trait-improving recombinant DNA can also be used toprovide transgenic plants having improved growth and development, andultimately increased yield, as the result of modified expression ofplant growth regulators or modification of cell cycle or photosynthesispathways.

“Expression” means transcription of DNA to produce RNA. The resultingRNA may be without limitation mRNA encoding a protein, antisense RNA, ora double-stranded RNA for use in RNAi technology. Expression also refersto production of encoded protein from mRNA.

A “plant promoter” is a promoter capable of initiating transcription inplant cells whether or not its origin is a plant cell. Exemplary plantpromoters include, but are not limited to, those that are obtained fromplants, plant viruses, and bacteria which comprise genes expressed inplant cells such Agrobacterium or Rhizobium. Examples of promoters underdevelopmental control include promoters that preferentially initiatetranscription in certain tissues, such as leaves, roots, or seeds. Suchpromoters are referred to as “tissue preferred”. Promoters whichinitiate transcription only in certain tissues are referred to as“tissue specific”. A “cell type” specific promoter primarily drivesexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. An “inducible” or “repressible”promoter is a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions, or certain chemicals, or thepresence of light. Tissue specific, tissue preferred, cell typespecific, and inducible promoters constitute the class of“non-constitutive” promoters. A “constitutive” promoter is a promoterwhich is active under most conditions. As used herein, “antisenseorientation” includes reference to a polynucleotide sequence that isoperably linked to a promoter in an orientation where the antisensestrand is transcribed. The antisense strand is sufficientlycomplementary to an endogenous transcription product such thattranslation of the endogenous transcription product is often inhibited.

As used herein, “operably linked” refers to the association of two ormore nucleic acid fragments on a single nucleic acid fragment so thatthe function of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

A “consensus sequence” refers to an artificial, amino acid sequence ofconserved parts of the proteins encoded by homologous genes, e.g., asdetermined by a CLUSTALW alignment of amino acid sequence of homologproteins.

Homologous genes are genes which encode proteins with the same orsimilar biological function to the protein encoded by the second gene.Homologous genes may be generated by the event of speciation (seeortholog) or by the event of genetic duplication (see paralog).“Orthologs” refer to a set of homologous genes in different species thatevolved from a common ancestral gene by specification. Normally,orthologs retain the same function in the course of evolution; and“paralogs” refer to a set of homologous genes in the same species thathave diverged from each other as a consequence of genetic duplication.Thus, homologous genes can be from the same or a different organism. Asused herein, “homolog” means a protein that performs the same biologicalfunction as a second protein including those identified by sequenceidentity search.

Percent identity refers to the extent to which two optimally aligned DNAor protein segments are invariant throughout a window of alignment ofcomponents, e.g., nucleotide sequence or amino acid sequence. An“identity fraction” for aligned segments of a test sequence and areference sequence is the number of identical components which areshared by sequences of the two aligned segments divided by the totalnumber of sequence components in the reference segment over a window ofalignment which is the smaller of the full test sequence or the fullreference sequence. “Percent identity” (“% identity”) is the identityfraction times 100. “% identity to a consensus amino acid sequence” is100 times the identity fraction in a window of alignment of an aminoacid sequence of a test protein optimally aligned to consensus aminoacid sequence of this invention.

“Arabidopsis” means plants of Arabidopsis thaliana.

“Pfam” refers to a large collection of multiple sequence alignments andhidden Markov models covering many common protein families, e.g. Pfamversion 18.0 (August 2005) contains alignments and models for 7973protein families and is based on the Swissprot 47.0 and SP-TREMBL 30.0protein sequence databases. See S. R. Eddy, “Profile Hidden MarkovModels”, Bioinformatics 14:755-763, 1998. Pfam is currently maintainedand updated by a Pfam Consortium. The alignments represent someevolutionary conserved structure that has implications for the protein'sfunction. Profile hidden Markov models (profile HMMs) built from thePfam alignments are useful for automatically recognizing that a newprotein belongs to an existing protein family even if the homology byalignment appears to be low. Once one DNA is identified as encoding aprotein which imparts an enhanced trait when expressed in transgenicplants, other DNA encoding proteins in the same protein family areidentified by querying the amino acid sequence of protein encoded bycandidate DNA against the Hidden Markov Model which characterizes thePfam domain using HMMER software, a current version of which is providedin the appended computer listing. Candidate proteins meeting thegathering cutoff for the alignment of a particular Pfam are in theprotein family and have cognate DNA that is useful in constructingrecombinant DNA for the use in the plant cells of this invention. HiddenMarkov Model databases for use with HMMER software in identifying DNAexpressing protein in a common Pfam for recombinant DNA in the plantcells of this invention are also included in the appended computerlisting. The HMMER software and Pfam databases are version 18.0 and wereused to identify known domains in the proteins corresponding to aminoacid sequence of SEQ ID NO: 198 through SEQ ID NO: 394. All DNA encodingproteins that have scores higher than the gathering cutoff disclosed inTable 17 by Pfam analysis disclosed herein can be used in recombinantDNA of the plant cells of this invention, e.g. for selecting transgenicplants having enhanced agronomic traits. The relevant Pfams for use inthis invention, as more specifically disclosed below, are L51_S25_CI-B8,iPGM_N, WD40, BPL_LipA_LipB, DUF676, AAA, S_locus_glycop, ArfGap,Rotamase, Metallophos, CMAS, Sugar_tr, LMBR1, RrnaAD, NAF, BolA,Pkinase, C2, FA_hydroxylase, p450, Complex1_30kDa, Histone, DUF822,PEP-utilizers, PCI, ETC_C1_NDUFA5, 2-Hacid_dh, Tryp_alpha_amyl, PK_C,MAP65_ASE1, FBPase, SWIB, Ank, Ribosomal_S8e, 2-Hacid_dh_C, SMC_N,GTP_cyclohydro2, PfkB, ORMDL, ADH_zinc_N, SWIM, TrkA_N, HLH, GH3, SNF5,Ceramidase_alk, Ribonuclease_T2, Complex1_49kDa, Gp_dh_C, Aldo_ket_red,zf-AN1, TFIIS_C, MFS_1, Thioredoxin, DUF1005, LEA_3, Sterol_MT_C,Gp_dh_N, TFIIS_M, PAN_2, BPL_C, DUF26, Aa_trans, ACT, ADH_N,NAD_binding_1, Auxin_inducible, B_lectin, Anti-silence, Response_reg,14-3-3, LRRNT_2, GDC-P, zf-CCHC, NPH3, TPR_1, TFIIA, DHBP_synthase,UQ_con, TPR_2, TPT, F-box, adh_short, Cyclin_C, Na_H_Exchanger,AA_permease, MtN3_slv, TIM, NDK, Pantoate_transf, Allene_ox_cyc,Cyclin_N, Methyltransf_11, CBM_20, Methyltransf_12, Rhodanese,Glycolytic, Actin, Usp, elF-4B, Glyco_transf_8, BURP, Alpha-amylase,F420_oxidored, EGF_CA, Kelch_1, PGAM, Aminotran_1_2, Kelch_2, UPF0261,CoA_binding, DUF868, Peptidase_S10, Lung_7-TM_R, Oleosin, Sad1_UNC,Gln-synt_C, LSM, NTP_transferase, Metalloenzyme, Prenylcys_lyase,Subtilisin_N, SAM_1, DUF298, ESCRT-III, DNA_pol_E_B, Aminotran_3,NAD_Gly3P_dh_N, Gln-synt_N, MMR_HSR1, DUF588, zf-CCCH, DnaJ,Pkinase_Tyr, Cupin_2, LRR_1, Cupin_3, zf-CSL, FART, HD, FH2, APC8, PTR2,MannoseP_isomer, Rib_5-P_isom_A, DUF1336, Phosphorylase, DUF1191, Asp,Mit_rib_S27, PAP_fibrillin, DUF1195, Aldedh, zf-C3HC4, PPR, PK, PurA,RMMBL, HTH_11, Tim17, and PBD.

Recombinant DNA Constructs

The present invention provides recombinant DNA constructs comprising oneor more polynucleotides disclosed herein for imparting one or moreimproved traits to transgenic plant when incorporated into the nucleusof the plant cells. Such constructs also typically comprise a promoteroperatively linked to said polynucleotide to provide for expression inthe plant cells. Other construct components may include additionalregulatory elements, such as 5′ or 3′ untranslated regions (such aspolyadenylation sites), intron regions, and transit or signal peptides.Such recombinant DNA constructs can be assembled using methods known tothose of ordinary skill in the art.

In a preferred embodiment, a polynucleotide of the present invention isoperatively linked in a recombinant DNA construct to a promoterfunctional in a plant to provide for expression of the polynucleotide inthe sense orientation such that a desired protein or polypeptidefragment of a protein is produced. Also provided are embodiments whereina polynucleotide is operatively linked to a promoter functional in aplant to provide for expression of gene suppression RNA to suppress thelevel of an endogenous protein.

Recombinant constructs prepared in accordance with the present inventionalso generally include a 3′ untranslated DNA region (UTR) that typicallycontains a polyadenylation sequence following the polynucleotide codingregion. Examples of useful 3′ UTRs include those from the nopalinesynthase gene of Agrobacterium tumefaciens (nos), a gene encoding thesmall subunit of a ribulose-1,5-bisphosphate carboxylase-oxygenase(rbcS), and the T7 transcript of Agrobacterium tumefaciens.

Constructs and vectors may also include a transit peptide for targetingof a gene target to a plant organelle, particularly to a chloroplast,leucoplast or other plastid organelle. For descriptions of the use ofchloroplast transit peptides, see U.S. Pat. No. 5,188,642 and U.S. Pat.No. 5,728,925, incorporated herein by reference.

Table 1 provides a list of genes that provided recombinant DNA that wasexpressed in a model plant and identified from screening as imparting animproved trait. When the stated orientation is “sense”, the expressionof the gene or a homolog in a crop plant provides the means to identifytransgenic events that provide an enhanced trait in the crop plant. Whenthe stated orientation is “antisense”, the suppression of the nativehomolog in a crop plant provides the means to identify transgenic eventsthat provide an enhanced trait in the crop plant. In some cases theexpression/suppression in the model plant exhibited an improved traitthat corresponds to an enhanced agronomic trait, e.g. cold stresstolerance, water deficit stress tolerance, low nitrogen stress toleranceand the like. In other cases the expression/suppression in the modelplant exhibited an improved trait that is a surrogate to an enhanceagronomic trait, e.g. salinity stress tolerance being a surrogate todrought tolerance or improvement in plant growth and development being asurrogate to enhanced yield. Even when expression of a transgene orsuppression of a native gene imparts an improved trait in a model plant,not every crop plant expressing the same transgene or suppressing thesame native gene will necessarily demonstrate an indicated enhancedagronomic trait. For instance, it is well known that multiple transgenicevents are required to identify a transgenic plant that can exhibit anenhanced agronomic trait. However, by with routine experimentation atransgenic plant cell nuclei, cell, plant or seed of this invention canbe identified by making a reasonable number of transgenic events andengaging in screening process identified in this specification andillustrated in the examples. An understanding of Table 1 is facilitatedby the following description of the headings:

“NUC SEQ ID NO” refers to a SEQ ID NO. for particular DNA sequence inthe Sequence Listing.

“PEP SEQ ID NO” refers to a SEQ ID NO. in the Sequence Listing for theamino acid sequence of a protein cognate to a particular DNA

“construct_id” refers to an arbitrary number used to identify aparticular recombinant DNA construct comprising the particular DNA.

“Gene ID” refers to an arbitrary name used to identify the particularDNA.

“orientation” refers to the orientation of the particular DNA in arecombinant DNA construct relative to the promoter.

TABLE 1 NUC PEP Seq SEQ ID ID Construct No. No Gene ID ID Orientation 1198 CGPG106 11029 SENSE 2 199 CGPG1133 12223 SENSE 3 200 CGPG117 10422ANTI-SENSE 4 201 CGPG1226 13485 ANTI-SENSE 5 202 CGPG1288 13235 SENSE 6203 CGPG1301 13411 SENSE 7 204 CGPG1458 73944 SENSE 8 205 CGPG1542 13846ANTI-SENSE 9 206 CGPG170 12602 SENSE 10 207 CGPG1828 74065 ANTI-SENSE 11208 CGPG2206 72783 SENSE 12 209 CGPG2217 17210 SENSE 13 210 CGPG229272724 SENSE 14 211 CGPG2457 17805 ANTI-SENSE 15 212 CGPG2499 16610 SENSE16 213 CGPG2653 76602 SENSE 17 214 CGPG2813 18456 SENSE 18 215 CGPG300218414 SENSE 19 216 CGPG3154 71538 SENSE 20 217 CGPG3235 76532 SENSE 21218 CGPG3274 18231 SENSE 22 219 CGPG3275 18232 SENSE 23 220 CGPG336318256 SENSE 24 221 CGPG3367 18258 SENSE 25 222 CGPG3375 19193 SENSE 26223 CGPG3528 71301 SENSE 27 224 CGPG3534 18354 SENSE 28 225 CGPG363877334 SENSE 29 226 CGPG3918 19767 SENSE 30 227 CGPG3920 19774 SENSE 31228 CGPG3962 70992 SENSE 32 229 CGPG3972 19956 SENSE 33 230 CGPG399070948 SENSE 34 231 CGPG3994 70201 SENSE 35 232 CGPG4026 19973 SENSE 36233 CGPG4048 70987 SENSE 37 234 CGPG4052 70950 SENSE 38 235 CGPG405770962 SENSE 39 236 CGPG4058 70915 SENSE 40 237 CGPG4069 19947 SENSE 41238 CGPG4087 70969 SENSE 42 239 CGPG4088 70985 SENSE 43 240 CGPG410270971 SENSE 44 241 CGPG4121 70963 SENSE 45 242 CGPG4122 70994 SENSE 46243 CGPG4140 70956 SENSE 47 244 CGPG4154 70995 SENSE 48 245 CGPG431173306 SENSE 49 246 CGPG4363 70657 SENSE 50 247 CGPG4369 70660 SENSE 51248 CGPG442 74536 SENSE 52 249 CGPG4454 71328 SENSE 53 250 CGPG445671329 SENSE 54 251 CGPG4473 70755 SENSE 55 252 CGPG4588 70684 SENSE 56253 CGPG4765 73330 SENSE 57 254 CGPG4788 76202 SENSE 58 255 CGPG491272807 SENSE 59 256 CGPG4926 72811 SENSE 60 257 CGPG4967 73235 SENSE 61258 CGPG4977 72813 SENSE 62 259 CGPG5001 72825 SENSE 63 260 CGPG502573628 SENSE 64 261 CGPG5041 76105 SENSE 65 262 CGPG5116 73242 SENSE 66263 CGPG5144 74217 SENSE 67 264 CGPG5171 73735 SENSE 68 265 CGPG519473256 SENSE 69 266 CGPG5200 73260 SENSE 70 267 CGPG5210 75822 SENSE 71268 CGPG5221 72001 SENSE 72 269 CGPG5269 72056 SENSE 73 270 CGPG540477308 SENSE 74 271 CGPG5432 73766 SENSE 75 272 CGPG5518 72774 SENSE 76273 CGPG5535 72788 SENSE 77 274 CGPG5540 72753 SENSE 78 275 CGPG556872709 SENSE 79 276 CGPG5577 73954 SENSE 80 277 CGPG5587 73137 SENSE 81278 CGPG5594 73161 SENSE 82 279 CGPG5633 73057 SENSE 83 280 CGPG564073127 SENSE 84 281 CGPG5646 73033 SENSE 85 282 CGPG5656 73105 SENSE 86283 CGPG5659 73141 SENSE 87 284 CGPG5661 73165 SENSE 88 285 CGPG568473155 SENSE 89 286 CGPG5694 73026 SENSE 90 287 CGPG5704 73120 SENSE 91288 CGPG5714 73133 SENSE 92 289 CGPG5721 73134 SENSE 93 290 CGPG572873123 SENSE 94 291 CGPG5757 73981 SENSE 95 292 CGPG5764 73136 SENSE 96293 CGPG5783 73172 SENSE 97 294 CGPG5791 73020 SENSE 98 295 CGPG579972946 SENSE 99 296 CGPG5856 74746 SENSE 100 297 CGPG5927 77312 SENSE 101298 CGPG5941 75237 SENSE 102 299 CGPG5957 75240 SENSE 103 300 CGPG596774349 SENSE 104 301 CGPG6040 76422 SENSE 105 302 CGPG607 70812 SENSE 106303 CGPG6178 77322 SENSE 107 304 CGPG6185 74662 SENSE 108 305 CGPG630676527 SENSE 109 306 CGPG6318 77020 SENSE 110 307 CGPG6326 77609 SENSE111 308 CGPG6370 73485 SENSE 112 309 CGPG6429 73433 SENSE 113 310CGPG6440 73411 SENSE 114 311 CGPG6516 73568 SENSE 115 312 CGPG6653 74688SENSE 116 313 CGPG6712 74420 SENSE 117 314 CGPG6737 74435 SENSE 118 315CGPG6747 74460 SENSE 119 316 CGPG6796 74566 SENSE 120 317 CGPG6805 77610SENSE 121 318 CGPG6810 77618 SENSE 122 319 CGPG6952 77517 SENSE 123 320CGPG6953 77518 SENSE 124 321 CGPG7121 76460 SENSE 125 322 CGPG7163 77069SENSE 126 323 CGPG7168 76161 SENSE 127 324 CGPG7206 76171 SENSE 128 325CGPG7225 76178 SENSE 129 326 CGPG7267 76467 SENSE 130 327 CGPG7272 77536SENSE 131 328 CGPG7281 76576 SENSE 132 329 CGPG7308 74862 SENSE 133 330CGPG7316 74863 SENSE 134 331 CGPG7371 74858 SENSE 135 332 CGPG7457 74933SENSE 136 333 CGPG7520 75379 SENSE 137 334 CGPG7529 77816 SENSE 138 335CGPG7636 75434 SENSE 139 336 CGPG7737 77821 SENSE 140 337 CGPG7767 75685SENSE 141 338 CGPG7804 75654 SENSE 142 339 CGPG7823 75692 SENSE 143 340CGPG7828 75657 SENSE 144 341 CGPG7833 75622 SENSE 145 342 CGPG7933 77549SENSE 146 343 CGPG7986 77917 SENSE 147 344 CGPG8012 77568 SENSE 148 345CGPG8015 77570 SENSE 149 346 CGPG8055 77338 SENSE 150 347 CGPG8062 77580SENSE 151 348 CGPG8082 77928 SENSE 152 349 CGPG8083 77349 SENSE 153 350CGPG8106 77357 SENSE 154 351 CGPG8107 77587 SENSE 155 352 CGPG8136 77933SENSE 156 353 CGPG8152 77619 SENSE 157 354 CGPG8166 77621 SENSE 158 355CGPG8377 77629 SENSE 159 356 CGPG8976 77832 SENSE 160 357 CGPG8987 76802SENSE 161 358 CGPG9013 76829 SENSE 162 359 CGPG9080 76961 SENSE 163 360CGPG9081 76973 SENSE 164 361 CGPG9130 77150 SENSE 165 362 CGPG9133 77186SENSE 166 363 CGPG9134 77103 SENSE 167 364 CGPG9137 77139 SENSE 168 365CGPG9141 77187 SENSE 169 366 CGPG9145 77140 SENSE 170 367 CGPG9147 77164SENSE 171 368 CGPG9148 77176 SENSE 172 369 CGPG9155 77165 SENSE 173 370CGPG9163 77166 SENSE 174 371 CGPG9170 77155 SENSE 175 372 CGPG9180 77180SENSE 176 373 CGPG9183 77121 SENSE 177 374 CGPG9186 77157 SENSE 178 375CGPG9205 77195 SENSE 179 376 CGPG9207 77124 SENSE 180 377 CGPG9219 77261SENSE 181 378 CGPG9220 77273 SENSE 182 379 CGPG9230 77203 SENSE 183 380CGPG9236 77275 SENSE 184 381 CGPG9238 77204 SENSE 185 382 CGPG9259 77266SENSE 186 383 CGPG9271 77220 SENSE 187 384 CGPG9275 77268 SENSE 188 385CGPG9278 77209 SENSE 189 386 CGPG9283 77269 SENSE 190 387 CGPG9309 77451SENSE 191 388 CGPG9311 77452 SENSE 192 389 CGPG9322 77430 SENSE 193 390CGPG9335 77432 SENSE 194 391 CGPG9341 77433 SENSE 195 392 CGPG9344 77444SENSE 196 393 CGPG9345 77409 SENSE 197 394 CGPG976 12313 ANTI- SENSE 10207 CGPG1828 16322 SENSE

Recombinant DNA

DNA for use in the present invention to improve traits in plants have anucleotide sequence of SEQ ID NO:1 through SEQ ID NO:197, as well as thehomologs of such DNA molecules. A subset of the DNA for gene suppressionaspects of the invention includes fragments of the disclosed fullpolynucleotides consisting of oligonucleotides of 21 or more consecutivenucleotides. Oligonucleotides the larger molecules having a sequenceselected from the group consisting of SEQ ID NO:1 through SEQ ID NO:197are useful as probes and primers for detection of the polynucleotidesused in the invention. Also useful in this invention are variants of theDNA. Such variants may be naturally occurring, including DNA fromhomologous genes from the same or a different species, or may benon-natural variants, for example DNA synthesized using chemicalsynthesis methods, or generated using recombinant DNA techniques.Degeneracy of the genetic code provides the possibility to substitute atleast one base of the protein encoding sequence of a gene with adifferent base without causing the amino acid sequence of thepolypeptide produced from the gene to be changed. Hence, a DNA useful inthe present invention may have any base sequence that has been changedfrom the sequences provided herein by substitution in accordance withdegeneracy of the genetic code.

Homologs of the genes providing DNA demonstrated as useful in improvingtraits in model plants disclosed herein will generally have significantidentity with the DNA disclosed herein. DNA is substantially identicalto a reference DNA if, when the sequences of the polynucleotides areoptimally aligned there is about 60% nucleotide equivalence; morepreferably 70%; more preferably 80% equivalence; more preferably 85%equivalence; more preferably 90%; more preferably 95%; and/or morepreferably 98% or 99% equivalence over a comparison window. A comparisonwindow is preferably at least 50-100 nucleotides, and more preferably isthe entire length of the polynucleotide provided herein. Optimalalignment of sequences for aligning a comparison window may be conductedby algorithms; preferably by computerized implementations of thesealgorithms (for example, the Wisconsin Genetics Software Package Release7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). Thereference polynucleotide may be a full-length molecule or a portion of alonger molecule. Preferentially, the window of comparison fordetermining polynucleotide identity of protein encoding sequences is theentire coding region.

Proteins useful for imparting improved traits are entire proteins or atleast a sufficient portion of the entire protein to impart the relevantbiological activity of the protein. Proteins useful for generation oftransgenic plants having improved traits include the proteins with anamino acid sequence provided herein as SEQ ID NO: 198 through SEQ ID NO:394, as well as homologs of such proteins.

Homologs of the proteins useful in the invention are identified bycomparison of the amino acid sequence of the protein to amino acidsequences of proteins from the same or different plant sources, e.g.,manually or by using known homology-based search algorithms such asthose commonly known and referred to as BLAST, FASTA, andSmith-Waterman. As used herein, a homolog is a protein from the same ora different organism that performs the same biological function as thepolypeptide to which it is compared. An orthologous relation between twoorganisms is not necessarily manifest as a one-to-one correspondencebetween two genes, because a gene can be duplicated or deleted afterorganism phylogenetic separation, such as speciation. For a givenprotein, there may be no ortholog or more than one ortholog. Othercomplicating factors include alternatively spliced transcripts from thesame gene, limited gene identification, redundant copies of the samegene with different sequence lengths or corrected sequence. A localsequence alignment program, e.g., BLAST, can be used to search adatabase of sequences to find similar sequences, and the summaryExpectation value (E-value) used to measure the sequence basesimilarity. As a protein hit with the best E-value for a particularorganism may not necessarily be an ortholog or the only ortholog, areciprocal BLAST search is used in the present invention to filter hitsequences with significant E-values for ortholog identification. Thereciprocal BLAST entails search of the significant hits against adatabase of amino acid sequences from the base organism that are similarto the sequence of the query protein. A hit is a likely ortholog, whenthe reciprocal BLAST's best hit is the query protein itself or a proteinencoded by a duplicated gene after speciation. Thus, homolog is usedherein to describe proteins that are assumed to have functionalsimilarity by inference from sequence base similarity. The relationshipof homologs with amino acid sequences of SEQ ID NO: 395 to SEQ ID NO:19,938 to the proteins with amino acid sequences of SEQ ID NO: to 198 toSEQ ID NO: 394 are found in the listing of Table 2.

Other functional homolog proteins differ in one or more amino acids fromthose of a trait-improving protein disclosed herein as the result of oneor more of the well-known conservative amino acid substitutions, e.g.,valine is a conservative substitute for alanine and threonine is aconservative substitute for serine. Conservative substitutions for anamino acid within the native sequence can be selected from other membersof a class to which the naturally occurring amino acid belongs.Representative amino acids within these various classes include, but arenot limited to: (1) acidic (negatively charged) amino acids such asaspartic acid and glutamic acid; (2) basic (positively charged) aminoacids such as arginine, histidine, and lysine; (3) neutral polar aminoacids such as glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) aminoacids such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine. Conserved substitutes for anamino acid within a native amino acid sequence can be selected fromother members of the group to which the naturally occurring amino acidbelongs. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Naturally conservative amino acids substitution groupsare: valine-leucine, valine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, aspartic acid-glutamic acid, andasparagine-glutamine. A further aspect of the invention comprisesproteins that differ in one or more amino acids from those of adescribed protein sequence as the result of deletion or insertion of oneor more amino acids in a native sequence.

Homologs of the trait-improving proteins provided herein will generallydemonstrate significant sequence identity. Of particular interest areproteins having at least 50% sequence identity, more preferably at leastabout 70% sequence identity or higher, e.g., at least about 80% sequenceidentity with an amino acid sequence of SEQ ID NO:198 through SEQ ID NO:394. Of course useful proteins also include those with higher identity,e.g., 90% to 99% identity. Identity of protein homologs is determined byoptimally aligning the amino acid sequence of a putative protein homologwith a defined amino acid sequence and by calculating the percentage ofidentical and conservatively substituted amino acids over the window ofcomparison. The window of comparison for determining identity can be theentire amino acid sequence disclosed herein, e.g., the full sequence ofany of SEQ ID NO: 198 through SEQ ID NO: 394.

Genes that are homologous to each other can be grouped into families andincluded in multiple sequence alignments. Then a consensus sequence foreach group can be derived. This analysis enables the derivation ofconserved and class-(family) specific residues or motifs that arefunctionally important. These conserved residues and motifs can befurther validated with 3D protein structure if available. The consensussequence can be used to define the full scope of the invention, e.g., toidentify proteins with a homolog relationship. Thus, the presentinvention contemplates that protein homologs include proteins with anamino acid sequence that has at least 90% identity to such a consensusamino acid sequence sequences.

Promoters

Numerous promoters that are active in plant cells have been described inthe literature. These include promoters present in plant genomes as wellas promoters from other sources, including nopaline synthase (NOS)promoter and octopine synthase (OCS) promoters carried on tumor-inducingplasmids of Agrobacterium tumefaciens, caulimovirus promoters such asthe cauliflower mosaic virus or Figwort mosaic virus promoters. Forinstance, see U.S. Pat. No. 5,858,742 and 5,322,938 which discloseversions of the constitutive promoter derived from cauliflower mosaicvirus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a FigwortMosaic Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which disclosesa maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a riceactin promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1 promoter,U.S. Pat. No. 6,232,526 which discloses a maize A3 promoter, U.S. Pat.No. 6,177,611 which discloses constitutive maize promoters, U.S. Pat.No. 6,433,252 which discloses a maize L3 oleosin promoter, U.S. Pat. No.6,429,357 which discloses a rice actin 2 promoter and intron, U.S. Pat.No. 5,837,848 which discloses a root specific promoter, U.S. Pat. No.6,084,089 which discloses cold inducible promoters, U.S. Pat. No.6,294,714 which discloses light inducible promoters, U.S. Pat. No.6,140,078 which discloses salt inducible promoters, U.S. Pat. No.6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No.6,175,060 which discloses phosphorus deficiency inducible promoters,U.S. Patent Application Publication 2002/0192813A1 which discloses 5′,3′ and intron elements useful in the design of effective plantexpression vectors, U.S. patent application Ser. No. 09/078,972 whichdiscloses a coixin promoter, U.S. patent application Ser. No. 09/757,089which discloses a maize chloroplast aldolase promoter, and U.S. patentapplication Ser. No. 10/739,565 which discloses water-deficit induciblepromoters, all of which are incorporated herein by reference. These andnumerous other promoters that function in plant cells are known to thoseskilled in the art and available for use in recombinant polynucleotidesof the present invention to provide for expression of desired genes intransgenic plant cells.

Furthermore, the promoters can include multiple “enhancer sequences” toassist in elevating gene expression. Such enhancers are known in theart. By including an enhancer sequence with such constructs, theexpression of the selected protein may be enhanced. These enhancersoften are found 5′ to the start of transcription in a promoter thatfunctions in eukaryotic cells, but can often be inserted in the forwardor reverse orientation 5′ or 3′ to the coding sequence. In someinstances, these 5′ enhancing elements are introns. Deemed to beparticularly useful as enhancers are the 5′ introns of the rice actin 1and rice actin 2 genes. Examples of other enhancers that can be used inaccordance with the invention include elements from the CaMV 35Spromoter, octopine synthase genes, the maize alcohol dehydrogenase gene,the maize shrunken 1 gene and promoters from non-plant eukaryotes.

In some aspects of the invention it is preferred that the promoterelement in the DNA construct be capable of causing sufficient expressionto result in the production of an effective amount of a polypeptide inwater deficit conditions. Such promoters can be identified and isolatedfrom the regulatory region of plant genes that are over expressed inwater deficit conditions. Specific water-deficit-inducible promoters foruse in this invention are derived from the 5′ regulatory region of genesidentified as a heat shock protein 17.5 gene (HSP 17.5), an HVA22 gene(HVA22), a Rab 17 gene and a cinnamic acid 4-hydroxylase (CA4H) gene(CA4H) of Zea maize. Such water-deficit-inducible promoters aredisclosed in U.S. application Ser. No.10/739,565, incorporated herein byreference.

In some aspects of the invention, sufficient expression in plant seedtissues is desired to effect improvements in seed composition. Exemplarypromoters for use for seed composition modification include promotersfrom seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al., (1997)Transgenic Res. 6(2):157-166), globulin 1 (Belanger et al., (1991)Genetics 129:863-872), glutelin 1 (Russell (1997) supra), andperoxiredoxin antioxidant (Per1) (Stacy et al., (1996) Plant Mol Biol.31(6):1205-1216).

In some aspects of the invention, preferential expression in plant greentissues is desired. Promoters of interest for such uses include thosefrom genes such as SSU (Fischhoff, et al., (1992) Plant Mol Biol.20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK)(Taniguchi, et al., (2000) Plant Cell Physiol. 41(1):42-48).

Gene suppression includes any of the well-known methods for suppressingtranscription of a gene or the accumulation of the mRNA corresponding tothat gene thereby preventing translation of the transcript into protein.Posttranscriptional gene suppression is mediated by transcription of RNAthat forms double-stranded RNA (dsRNA) having homology to a genetargeted for suppression. Suppression can also be achieved by insertionmutations created by transposable elements may also prevent genefunction. For example, in many dicot plants, transformation with theT-DNA of Agrobacterium may be readily achieved and large numbers oftransformants can be rapidly obtained. Also, some species have lineswith active transposable elements that can efficiently be used for thegeneration of large numbers of insertion mutations, while some otherspecies lack such options. Mutant plants produced by Agrobacterium ortransposon mutagenesis and having altered expression of a polypeptide ofinterest can be identified using the polynucleotides of the presentinvention. For example, a large population of mutated plants may bescreened with polynucleotides encoding the polypeptide of interest todetect mutated plants having an insertion in the gene encoding thepolypeptide of interest.

Gene Stacking

The present invention also contemplates that the trait-improvingrecombinant DNA provided herein can be used in combination with otherrecombinant DNA to create plants with multiple desired traits or afurther enhanced trait. The combinations generated can include multiplecopies of any one or more of the recombinant DNA constructs. Thesestacked combinations can be created by any method, including but notlimited to cross breeding of transgenic plants, or multiple genetictransformation.

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind to the proteinsdisclosed herein. Means for preparing and characterizing antibodies arewell known in the art (See, e.g., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference). Themethods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include using glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified antifungal protein, polypeptide or peptide. Theimmunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep, or frog cells isalso possible. The use of rats may provide certain advantages (Goding,1986, pp. 60-61), but mice are preferred, with the BALB/c mouse beingmost preferred as this is most routinely used and generally gives ahigher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986, pp. 65-66; Campbell, 1984, pp.75-83). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Spend virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically inducedfusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azasenne blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

Transformation Methods

Numerous methods for producing plant cell nuclei with recombinant DNAare known in the art and may be used in the present invention. Twocommonly used methods for plant transformation areAgrobacterium-mediated transformation and microprojectile bombardment.Microprojectile bombardment methods are illustrated in U.S. Pat. No.5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No.5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No.6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No.6,153,812 (wheat) and Agrobacterium-mediated transformation is describedin U.S. Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean);U.S. Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean),all of which are incorporated herein by reference. For Agrobacteriumtumefaciens based plant transformation system, additional elementspresent on transformation constructs will include T-DNA left and rightborder sequences to facilitate incorporation of the recombinantpolynucleotide into the plant genome.

In general it is preferred to introduce heterologous DNA randomly, i.e.,at a non-specific location, in the genome of a target plant line. Inspecial cases it may be useful to target heterologous DNA insertion inorder to achieve site-specific integration, e.g., to replace an existinggene in the genome, to use an existing promoter in the plant genome, orto insert a recombinant polynucleotide at a predetermined site known tobe active for gene expression. Several site specific recombinationsystems exist which are known to function implants include cre-lox asdisclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S.Pat. No. 5,527,695, both incorporated herein by reference.

Transformation methods of this invention are preferably practiced intissue culture on media and in a controlled environment. “Media” refersto the numerous nutrient mixtures that are used to grow cells in vitro,that is, outside of the intact living organism. Recipient cell targetsinclude, but are not limited to, meristem cells, callus, immatureembryos and gametic cells such as microspores, pollen, sperm and eggcells. It is contemplated that any cell from which a fertile plant maybe regenerated is useful as a recipient cell. Callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Cells capable ofproliferating as callus are also recipient cells for genetictransformation. Practical transformation methods and materials formaking transgenic plants of this invention, e.g., various media andrecipient target cells, transformation of immature embryos andsubsequent regeneration of fertile transgenic plants are disclosed inU.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. patent application Ser.No. 09/757,089, which are incorporated herein by reference.

In practice DNA is introduced into only a small percentage of targetcell nuclei in any one experiment. Marker genes are used to provide anefficient system for identification of those cells with nuclei that arestably transformed by receiving and integrating a transgenic DNAconstruct into their genomes. Preferred marker genes provide selectivemarkers that confer resistance to a selective agent, such as anantibiotic or herbicide. Potentially transformed cells with a nucleus ofthe invention are exposed to the selective agent. In the population ofsurviving cells will be those cells where, generally, theresistance-conferring gene has been integrated and expressed atsufficient levels to permit cell survival. Cells may be tested furtherto confirm stable integration of the exogenous DNA in the nucleus.Useful selective marker genes include those conferring resistance toantibiotics such as kanamycin (nptII), hygromycin B (aph IV) andgentamycin (aac3 and aacC4) or resistance to herbicides such asglufosinate (bar or pat) and glyphosate (EPSPS). Examples of suchselectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435;5,780,708 and 6,118,047, all of which are incorporated herein byreference. Screenable markers which provide an ability to visuallyidentify transformants can also be employed, e.g., a gene expressing acolored or fluorescent protein such as a luciferase or green fluorescentprotein (GFP) or a gene expressing a beta-glucuronidase or uidA gene(GUS) for which various chromogenic substrates are known. It is alsocontemplated that combinations of screenable and selectable markers willbe useful for identification of transformed cells. See PCT publicationWO 99/61129 which discloses use of a gene fusion between a selectablemarker gene and a screenable marker gene, e.g., an NPTII gene and a GFPgene.

Cells that survive exposure to the selective agent, or cells that havebeen scored positive in a screening assay, may be cultured inregeneration media and allowed to mature into plants. Developingplantlets can be transferred to soil less plant growth mix, and hardenedoff, e.g., in an environmentally controlled chamber at about 85%relative humidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻²s⁻¹ oflight, prior to transfer to a greenhouse or growth chamber formaturation. Plants are preferably matured either in a growth chamber orgreenhouse. Plants are regenerated from about 6 wk to 10 months after atransformant is identified, depending on the initial tissue. Duringregeneration, cells are grown to plants on solid media at about 19 to28° C. After regenerating plants have reached the stage of shoot androot development, they may be transferred to a greenhouse for furthergrowth and testing. Plants may be pollinated using conventional plantbreeding methods known to those of skill in the art and seed produced.

Progeny may be recovered from transformed plants and tested forexpression of the exogenous recombinant polynucleotide. Useful assaysinclude, for example, “molecular biological” assays, such as Southernand Northern blotting and PCR; “biochemical” assays, such as detectingthe presence of RNA, e.g., double stranded RNA, or a protein product,e.g., by immunological means (ELISAs and Western blots) or by enzymaticfunction; plant part assays, such as leaf or root assays; and also, byanalyzing the phenotype of the whole regenerated plant.

Discovery of Trait-Improving Recombinant DNA

To identify nuclei with recombinant DNA that confer improved traits toplants, Arabidopsis thaliana was transformed with a candidaterecombinant DNA construct and screened for an improved trait.

Arabidopsis thaliana is used a model for genetics and metabolism inplants. Arabidopsis has a small genome, and well-documented studies areavailable. It is easy to grow in large numbers and mutants definingimportant genetically controlled mechanisms are either available, or canreadily be obtained. Various methods to introduce and express isolatedhomologous genes are available (see Koncz, e.g., Methods in ArabidopsisResearch e.g., (1992), World Scientific, New Jersey, N.J., in“Preface”).

A two-step screening process was employed which comprised two passes oftrait characterization to ensure that the trait modification wasdependent on expression of the recombinant DNA, but not due to thechromosomal location of the integration of the transgene. Twelveindependent transgenic lines for each recombinant DNA construct wereestablished and assayed for the transgene expression levels. Fivetransgenic lines with high transgene expression levels were used in thefirst pass screen to evaluate the transgene's function in T2 transgenicplants. Subsequently, three transgenic events, which had been shown tohave one or more improved traits, were further evaluated in the secondpass screen to confirm the transgene's ability to impart an improvedtrait. The following Table 3 summarizes the improved traits that havebeen confirmed as provided by a recombinant DNA construct.

In particular, Table 3 reports:

“PEP SEQ ID” which is the amino acid sequence of the protein cognate tothe DNA in the recombinant DNA construct corresponding to a proteinsequence of a SEQ ID NO. in the Sequence Listing.

“construct_id” is an arbitrary name for the recombinant DNA describemore particularly in Table 1.

“annotation” refers to a description of the top hit protein obtainedfrom an amino acid sequence query of each PEP SEQ ID NO to GenBankdatabase of the National Center for Biotechnology Information (ncbi).More particularly, “gi” is the GenBank ID number for the top BLAST hit.

“description” refers to the description of the top BLAST hit.

“e-value” provides the expectation value for the BLAST hit.

“% id” refers to the percentage of identically matched amino acidresidues along the length of the portion of the sequences which isaligned by BLAST between the sequence of interest provided herein andthe hit sequence in GenBank.

“traits” identify by two letter codes the confirmed improvement in atransgenic plant provided by the recombinant DNA. The codes for improvedtraits are:

“CK” which indicates cold tolerance improvement identified under a coldshock tolerance screen;

“CS” which indicates cold tolerance improvement identified by a coldgermination tolerance screen;

“DS” which indicates drought tolerance improvement identified by a soildrought stress tolerance screen;

“PEG” which indicates osmotic stress tolerance improvement identified bya PEG induced osmotic stress tolerance screen;

“HS” which indicates heat stress tolerance improvement identified by aheat stress tolerance screen;

“SS” which indicates high salinity stress tolerance improvementidentified by a salt stress tolerance screen;

“LN” which indicates nitrogen use efficiency improvement identified by alimited nitrogen tolerance screen; “LL” which indicates attenuated shadeavoidance response identified by a shade tolerance screen under a lowlight condition;

“PP” which indicates improved growth and development at early stagesidentified by an early plant growth and development screen;

“SP” which indicates improved growth and development at late stagesidentified by a late plant growth and development screen providedherein.

TABLE 3 PEP Annotation SEQ Construct e % ID ID value id descriptiontrait 198 11029 1.00E−170 85 gb|AAD02882.1|gamma-tocopherol DS LNmethyltransferase [Arabidopsis thaliana] 199 12223 / / / CK PEG 20010422 1.00E−133 100 gb|AAB47766.1|SNF5 homolog BSH LN [Arabidopsisthaliana] 201 13485 4.00E−39 55 emb|CAB79347.1|hypothetical LN protein[Arabidopsis thaliana] 202 13235 7.00E−76 100 emb|CAB78153.1|putativeprotein LN [Arabidopsis thaliana] 203 13411 3.00E−29 100ref|NP_179638.1|unknown protein LN CK [Arabidopsis thaliana] 204 73944 095 ref|NP_189578.1|phosphorylase/ CS transferase, transferring glycosylgroups [Arabidopsis thaliana] 205 13846 0 91 gb|AAN60291.1|unknown LN[Arabidopsis thaliana] 206 12602 0 100 ref|NP_565974.1|PP2A-4;hydrolase/ LN protein phosphatase type 2A/protein serine/threoninephosphatase [Arabidopsis thaliana] 207 74065 0 94 ref|NP_181390.1|DNAbinding/ CS DS transcription factor [Arabidopsis thaliana]gb|AAN13033.1|putative elongation 208 72783 0 83 ref|NP_564690.1|unknownprotein CK PEG HS [Arabidopsis thaliana] 209 17210 1.00E−92 100ref|NP_563622.1|unknown protein CS CK PEG [Arabidopsis thaliana] 21072724 1.00E−128 90 ref|NP_190525.1|protein translocase/ LL proteintransporter [Arabidopsis thaliana 211 17805 1.00E−172 80ref|NP_563761.1|unknown protein LN [Arabidopsis thaliana] 212 16610 0100 gb|AAL44550.1|fructose bisphosphate LN HS aldolase [Agrobacteriumtumefaciens str. C58] 213 76602 0 78 ref|NP_566515.1|signal transducerCS HS [Arabidopsis thaliana] 214 18456 1.00E−135 81 ref|NP_176820.1|DNAbinding/ CK SS PEG transcription factor [Arabidopsis thaliana] 215 184141.00E−169 70 ref|NP_195583.1|translation initiation CK factor[Arabidopsis thaliana] 216 71538 1.00E−137 90emb|CAE85115.1|synaptotagmin SP [Arabidopsis thaliana] 217 76532 0 99ref|NP_564775.1|ATP binding/ CK CS carbohydrate binding/kinase/proteinkinase/protein serine/threonine kinase/protein-tyrosine kinase/sugarbinding [Arabidopsis thaliana] 218 18231 1.00E−142 100emb|CAC83762.1|allene oxide CK LN cyclase [Arabidopsis thaliana] 21918232 6.00E−85 89 ref|NP_191628.1|unknown protein CS [Arabidopsisthaliana] 220 18256 1.00E−102 100 dbj|BAD44508.1|putative zinc finger SSCK HS protein (PMZ) [Arabidopsis thaliana] 221 18258 1.00E−175 83emb|CAA11525.1|transcription factor SP IIA large subunit [Arabidopsisthaliana] 222 19193 1.00E−103 100 ref|NP_564682.1|lipid binding SP HS[Arabidopsis thaliana] dbj|BAE73268.1|xylogen like protein 12[Arabidopsis thaliana] 223 71301 0 84 ref|NP_568393.1|nucleic acidbinding LN [Arabidopsis thaliana] 224 18354 0 96 ref|NP_567568.1|ATPbinding/ LN kinase/protein kinase/protein serine/threoninekinase/protein- tyrosine kinase [Arabidopsis thaliana] 225 773341.00E−117 100 ref|NP_178399.1|RNS1 LL (RIBONUCLEASE 1); endoribonuclease[Arabidopsis thaliana] 226 19767 1.00E−180 95 gb|AAO69665.1|serinethreonine CS DS protein phosphatase [Phaseolus acutifolius] 227 197741.00E−100 57 ref|NP_974271.1|unknown protein CS PP [Arabidopsisthaliana] 228 70992 1.00E−108 65 gb|ABE87200.1|hypothetical protein CSPEG MtrDRAFT_AC151668g4v1 [Medicago truncatula] 229 19956 1.00E−146 100gb|AAF64040.1|14-3-3-like protein PP [Glycine max] 230 70948 3.00E−59 45ref|NP_201297.1|CIP8 (COP1- PP HS INTERACTING PROTEIN 8); proteinbinding/zinc ion binding [Arabidopsis thaliana] 231 70201 0 68ref|NP_567072.1|ATP binding/ CS SS kinase/protein kinase/proteinserine/threonine kinase/protein- tyrosine kinase [Arabidopsis thaliana]232 19973 0 96 gb|AAB04057.1|S-adenosyl-L- PP SSmethionine:delta24-sterol-C- methyltransferase 233 70987 0 93gb|AAM83095.1|SOS2-like protein CK CS HS SS PEG kinase [Glycine max] 23470950 1.00E−116 77 dbj|BAD38167.1|putative leucine PP PEG zipper protein[Oryza sativa (japonica cultivar-group)] 235 70962 1.00E−179 78gb|ABE93200.1|conserved PP HS hypothetical protein [Medicago truncatula]236 70915 0 76 dbj|BAE71210.1|hypothetical protein CS PP [Trifoliumpratense] dbj|BAE71208.1| hypothetical protein [Trifolium pratense] 23719947 1.00E−116 65 gb|AAS38575.1|short-chain CS DS SS dehydrogenaseTic32 [Pisum sativum] 238 70969 1.00E−144 76 ref|NP_174321.2|unknownprotein SS [Arabidopsis thaliana] 239 70985 0 69 dbj|BAB08296.1|unnamedprotein CS HS PEG product [Arabidopsis thaliana] sp|Q9FMT4|Y5417_ARATHProtein At5g14170 240 70971 1.00E−92 63 gb|ABE80664.1|GNS1/SUR4 SSmembrane protein [Medicago truncatula] gb|ABE78502.1| GNS1/SUR4 membraneprotein [Medicago truncatula] 241 70963 1.00E−96 56ref|NP_177178.1|CYCD1; 1; cyclin- CS PP HS dependent protein kinaseregulator [Arabidopsis thaliana] 242 70994 2.00E−33 44ref|NP_172962.1|RHA2A; protein PP DS HS SS binding/ubiquitin-proteinligase/zinc ion binding [Arabidopsis thaliana] 243 70956 0 85gb|AAO23063.1|ent-kaurenoic acid CS SS HS oxidase [Pisum sativum] 24470995 1.00E−132 66 dbj|BAD73789.1|putative CK CS PP LL PEGuncharacterized hypothalamus protein HT010 [Oryza sativa (japonicacultivar-group)] 245 73306 0 93 ref|NP_201243.1|phosphoric ester SS PEGhydrolase [Arabidopsis thaliana] 246 70657 9.00E−31 73emb|CAB78150.1|probable wound- SP LL induced protein [Arabidopsisthaliana] 247 70660 2.00E−32 79 ref|NP_568217.1|transcription SPregulator [Arabidopsis thaliana] 248 74536 0 91 ref|NP_001031061.1|PGDH;CS phosphoglycerate dehydrogenase [Arabidopsis thaliana]] 249 71328 0 99gb|AAD46022.1|Strong simlarity to PP gb|286426 F10M6.190 cytochrome p450homolog from Arabidopsis thaliana BAC 250 71329 0 94ref|NP_177477.1|heme binding/iron LN ion binding/monooxygenase/oxygenbinding [Arabidopsis thaliana] 251 70755 1.00E−142 100ref|NP_188925.1|unknown protein LL [Arabidopsis thaliana] 252 706843.00E−53 100 emb|CAB80194.1|putative protein CS LN HS PP [Arabidopsisthaliana] 253 73330 1.00E−158 72 emb|CAB96855.1|putative protein CK CSSS [Arabidopsis thaliana] 254 76202 3.00E−83 67 emb|CAA16586.1|putativeprotein LL [Arabidopsis thaliana] 255 72807 0 100ref|NP_173478.2|nucleotide binding PEG CK HS SS [Arabidopsis thaliana]256 72811 1.00E−176 100 ref|NP_565782.1|nucleotide binding CK SS PEG[Arabidopsis thaliana] 257 73235 0 89 ref|NP_564867.1|pepsin A SS HS[Arabidopsis thaliana] 258 72813 2.00E−79 93 ref|NP_850506.1|unknownprotein LL LN [Arabidopsis thaliana] 259 72825 1.00E−154 95ref|NP_563635.1|oxidoreductase CK PEG HS [Arabidopsis thaliana] 26073628 1.00E−163 92 ref|NP_849428.1|oxidoreductase CS DS LL HS PEG[Arabidopsis thaliana] 261 76105 0 93 emb|CAB79123.1|receptorkinase-like DS protein [Arabidopsis thaliana] 262 73242 0 100ref|NP_567292.1|unknown protein SP PEG [Arabidopsis thaliana] 263 742170 100 emb|CAB93726.1|cytochrome P450- DS CK like protein [Arabidopsisthaliana] 264 73735 0 97 gb|AAU94404.1|At3g48520 CK [Arabidopsisthaliana] 265 73256 1.00E−114 77 ref|NP_566329.1|unknown protein CS PPPEG [Arabidopsis thaliana] gb|AAL06975.1| 266 73260 4.00E−37 100ref|NP_566488.1|unknown protein CS [Arabidopsis thaliana] 267 758221.00E−90 88 emb|CAB63015.1|putative protein LL [Arabidopsis thaliana]268 72001 0 96 ref|NP_200045.1|CYP96A4; heme CS PP HS binding/iron ionbinding/ monooxygenase/oxygen binding [Arabidopsis thaliana] 269 72056 092 ref|NP_010186.1|Essential, non- SP ATPase regulatory subunit of the26S proteasome lid required for the assembly and activity of the 26Sproteasome; 270 77308 0 100 ref|NP_178642.1|SCPL38; serine PEGcarboxypeptidase [Arabidopsis thaliana] 271 73766 1.00E−163 100emb|CAB62363.1|MTN3-like protein LL [Arabidopsis thaliana] 272 727741.00E−139 95 ref|NP_011052.1|Constituent of 66S LL CK HS pre-ribosomalparticles, involved in 60S ribosomal subunit biogenesis; 273 72788 0 81ref|NP_010007.1|General repressor of CK CS SS transcription, formscomplex with Cyc8p, involved in the establishment of repressivechromatin structure through interactions with histones H3 and H4,appears to enhance expression of some genes 274 72753 1.00E−105 85ref|NP_013125.1|One of four subunits CS PP HS PEG of the endosomalsorting complex required for transport III (ESCRT- III); 275 72709 0 94ref|NP_011827.1|Low affinity LL methionine permease, similar to Mup1p;276 73954 0 98 ref|NP_200954.1|MIM; ATP binding HS [Arabidopsisthaliana] 277 73137 1.00E−110 94 gb|AAD32800.1|putative thioredoxin CS H[Arabidopsis thaliana] 278 73161 1.00E−36 65 emb|CAB85993.1|putativeprotein CS PEG [Arabidopsis thaliana] 279 73057 1.00E−133 95ref|NP_012770.1|Tetrameric SP phosphoglycerate mutase, mediates theconversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysisand the reverse reaction during gluconeogenesis; 280 73127 0 99dbj|BAB05414.1|aspartate SS HS aminotransferase [Bacillus haloduransC-125] ref|NP_242561.1|aspartate aminotransferase [Bacillus haloduransC-125] 281 73033 0 96 dbj|BAB07276.1|2,3- CKbisphosphoglycerate-independent phosphoglycerate mutase [Bacillushalodurans C-125] 282 73105 0 99 emb|CAB13630.1|glutamine PP PEGsynthetase [Bacillus subtilis subsp. subtilis str. 168] 283 73141 0 95ref|NP_414696.1|glutamate-1- CS semialdehyde aminotransferase[Escherichia coli K12] 284 73165 0 99 ref|NP_288110.1|pyruvate kinase DSPEG [Escherichia coli O157:H7 EDL933] 285 73155 1.00E−173 92emb|CAC47346.1|PROBABLE CS FRUCTOSE-BISPHOSPHATE ALDOLASE CLASS IPROTEIN [Sinorhizobium meliloti]] 286 73026 1.00E−116 87emb|CAG77169.1|triosephosphate PEG isomerase [Erwinia carotovora subsp.atroseptica SCRI1043] 287 73120 8.00E−82 100 ref|NP_441918.1|nucleosideCS SS HS diphosphate kinase [Synechocystis sp. PCC 6803] 288 731332.00E−63 90 gb|ABA76342.1|Nucleoside LL diphosphate kinase [Pseudomonasfluorescens PfO-1] 289 73134 9.00E−98 81 emb|CAE15979.1|ribose5-phosphate SS CK isomerase A (phosphoriboisomerase A) [Photorhabdusluminescens subsp. laumondii TTO1] 290 73123 0 95ref|NP_180412.2|nucleic acid binding CS PEG [Arabidopsis thaliana] 29173981 0 93 ref|NP_014797.1|hypothetical protein; DS PEG Slp1p[Saccharomyces cerevisiae] 292 73136 0 91 ref|NP_012454.1|Nuclear actin-DS PP related protein involved in chromatin remodeling 293 73172 0 100ref|NP_014952.1|Protein involved in CK PEG HS ER-to-Golgi transport;Sly41p [Saccharomyces cerevisiae] 294 73020 0 93 ref|NP_014993.1|Prolinepermease, LL required for high-affinity transport of proline 295 72946 095 ref|NP_012534.1|Vacuolar PEG LL transporter, imports large neutralamino acids into the vacuole 296 74746 0 93 ref|NP_566298.1|ATP binding/CK kinase/protein kinase/protein serine/threonine kinase/protein-tyrosine kinase [Arabidopsis thaliana] 297 77312 0 90ref|NP_178463.1|CDC48B; ATP PP SS HS binding/ATP-dependent peptidase/ATPase/nucleoside-triphosphatase/ nucleotide binding/serine-typeendopeptidase [Arabidopsis thaliana] 298 75237 0 100ref|NP_173390.1|kinase [Arabidopsis LN thaliana] gb|AAF98405.1|Unknownprotein [Arabidopsis thaliana] 299 75240 0 100 emb|CAB69854.1|putativeprotein CK [Arabidopsis thaliana] 300 74349 1.00E−148 79ref|NP_565187.1|transcription LN regulator [Arabidopsis thaliana] 30176422 5.00E−44 100 ref|NP_171781.1|unknown protein LN [Arabidopsisthaliana] 302 70812 1.00E−152 92 ref|NP_850182.1|PUR ALPHA-1; PP PEGnucleic acid binding [Arabidopsis thaliana] 303 77322 1.00E−172 100gb|AAW38983.1|At5g10750 LN LL [Arabidopsis thaliana] 304 74662 4.00E−89100 ref|NP_001031939.1|ubiquitin PP HS conjugating enzyme/ubiquitin-likeactivating enzyme [Arabidopsis thaliana] 305 76527 0 96emb|CAB51062.1|cell division cycle CS protein 23 homolog [Arabidopsisthaliana] 306 77020 0 98 emb|CAB75781.1|putative transporter CS protein[Arabidopsis thaliana] ref|NP_190154.1|transporter [Arabidopsisthaliana] 307 77609 0 96 gb|AAU95452.1|At5g04420 LL SS [Arabidopsisthaliana] 308 73485 0 100 emb|CAD84238.1|Glyceraldehyde 3- PEG phosphatedehydrogenase 309 73433 1.00E−132 65 ref|ZP_00819153.1|putative alcoholSP LN PP dehydrogenase [Marinobacter aquaeolei VT8] 310 73411 0 99emb|CAB14878.1|pyruvate kinase CS HS PP PEG [Bacillus subtilis subsp.subtilis str. 168] 311 73568 0 97 emb|CAC41401.1|PROBABLE SPSUCCINATE-SEMIALDEHYDE DEHYDROGENASE [NADP+] PROTEIN [Sinorhizobiummeliloti] 312 74688 0 98 emb|CAA16688.1|receptor protein CS PP SS PEGkinase - like protein [Arabidopsis thaliana] 313 74420 1.00E−142 99emb|CAD85691.1|Phosphoglycerate PP LN SS mutase family [Nitrosomonaseuropaea ATCC 19718] 314 74435 1.00E−117 100 emb|CAC41549.1|PROBABLE LLPHOSPHOGLYCERATE MUTASE 1 PROTEIN [Sinorhizobium meliloti] 315 74460 095 ref|NP_441738.1|fructose-1,6- CK bisphosphatase [Synechocystis sp.PCC 6803] 316 74566 0 93 emb|CAC48499.1|putative trehalose SP PP SS PEGHS synthase protein [Sinorhizobium meliloti 1021] 317 77610 0 94gb|AAC23406.1|hypothetical protein CS HS [Arabidopsis thaliana] 31877618 0 100 ref|NP_850453.1|JAR1 PP HS (JASMONATE RESISTANT 1)[Arabidopsis thaliana] 319 77517 7.00E−77 100 emb|CAB75802.1|putativeprotein CS LL [Arabidopsis thaliana] 320 77518 1.00E−49 100ref|NP_199600.1|oxidoreductase, SS LL HS acting on NADH or NADPH,quinone or similar compound as acceptor [Arabidopsis thaliana] 321 76460/ / / CS PEG 322 77069 0 85 ref|NP_201196.1|unknown protein PP[Arabidopsis thaliana] 323 76161 1.00E−161 100 ref|NP_851282.1|unknownprotein CK [Arabidopsis thaliana] 324 76171 1.00E−178 95ref|NP_176204.1|oxidoreductase LL [Arabidopsis thaliana] 325 761785.00E−60 86 ref|NP_565029.1|unknown protein CS PP HS LL PEG [Arabidopsisthaliana] 326 76467 6.00E−86 89 ref|NP_565671.1|unknown protein LL[Arabidopsis thaliana] 327 77536 1.00E−119 83 gb|AAS99692.1|At1g10020 SPCS DS PP LN [Arabidopsis thaliana] 328 76576 1.00E−145 100ref|NP_181023.1|FAH1 (FATTY DS ACID HYDROXYLASE 1); catalytic[Arabidopsis thaliana] 329 74862 1.00E−145 100 gb|ABA77057.1|Delta1-pyrroline-5- PP carboxylate reductase [Pseudomonas fluorescens PfO-1]330 74863 1.00E−93 100 ref|NP_012420.1|Nucleosome LL PEG CK SS assemblyfactor, involved in chromatin assembly after DNA replication] 331 748585.00E−50 68 gb|AAK14395.1|response regulator LL protein [Dianthuscaryophyllus] 332 74933 1.00E−177 95 gb|AAK85899.1|AGR_C_118p LL[Agrobacterium tumefaciens str. C58] 333 75379 1.00E−105 76dbj|BAD73205.1|unknown protein LN [Oryza sativa (japonica cultivar-group)] 334 77816 4.00E−41 89 gb|ABA98984.1|expressed protein SP PP HS[Oryza sativa (japonica cultivar- group)] 335 75434 3.00E−52 79ref|XP_472650.1|OSJNBa0027P08.15 LN [Oryza sativa (japonica cultivar-group)] 336 77821 0 71 gb|ABE84883.1|conserved PP SS HS PEG hypotheticalprotein [Medicago truncatula] 337 75685 0 82 ref|XP_475937.1|unknownprotein LL [Oryza sativa (japonica cultivar- group)] 338 75654 0 100ref|NP_563865.1|unknown protein SP CK HS [Arabidopsis thaliana] 33975692 1.00E−119 87 dbj|BAD30296.1|peptidyl-prolyl cis- CK LN transisomerase-like protein [Oryza sativa (japonica cultivar-group)] 34075657 6.00E−57 67 dbj|BAD38392.1|DNAJ heat shock N- CS LL HS terminaldomain-containing protein- like [Oryza sativa (japonica cultivar-group)] 341 75622 1.00E−30 78 dbj|BAD45825.1|unknown protein LN [Oryzasativa (japonica cultivar- group)] 342 77549 1.00E−132 89emb|CAB80891.1|AT4g00820 PP HS [Arabidopsis thaliana]] 343 77917 0 91ref|NP_197917.1|EBF2 (EIN3- PP PEG BINDING F BOX PROTEIN 2) [Arabidopsisthaliana] 344 77568 9.00E−99 76 ref|NP_849792.1|nucleic acid binding PP[Arabidopsis thaliana] 345 77570 7.00E−54 100 gb|AAS75309.1|multidomainCK SS cyclophilin type peptidyl-prolyl cis- trans isomerase [Arabidopsisthaliana]] 346 77338 4.00E−24 65 gb|AAM61454.1|unknown PEG [Arabidopsisthaliana] 347 77580 3.00E−45 100 dbj|BAB01457.1|unnamed protein PEG CSHS SS product [Arabidopsis thaliana] 348 77928 4.00E−38 100ref|NP_196244.1|unknown protein HS [Arabidopsis thaliana] 349 773491.00E−81 93 emb|CAC05463.1|putative lipid LL SS transfer protein[Arabidopsis thaliana] 350 77357 1.00E−155 95 ref|NP_175357.1|unknownprotein PEG [Arabidopsis thaliana] 351 77587 / / / CS PP HS SS 352 77933/ / / SS 353 77619 3.00E−40 98 dbj|BAB09403.1|unnamed protein CK SS PEGHS product [Arabidopsis thaliana] 354 77621 4.00E−58 74ref|NP_197632.1|Rac GTPase CK activator [Arabidopsis thaliana][Arabidopsis thaliana] 355 77629 5.00E−36 100 ref|NP_196372.1|GRP19[Arabidopsis SS HS thaliana] 356 77832 0 83 ref|NP_917762.1|P0501G01.24CS SS PEG HS LN [Oryza sativa (japonica cultivar- group)] 357 76802 0 76ref|NP_181908.1|actin binding CK [Arabidopsis thaliana]gb|AAB64026.1|unknown protein [Arabidopsis thaliana] 358 76829 0 89ref|NP_909912.1|ferredoxin-NADP+ CK reductase [Oryza sativa] 359 76961 065 ref|XP_473189.1|OSJNBa0073E02.11 LL LN [Oryza sativa (japonicacultivar- group)] 360 76973 1.00E−151 76 ref|XP_480055.1|unknown proteinLL [Oryza sativa (japonica cultivar- group)] 361 77150 0 76dbj|BAD61385.1|putative SP HS PEG nucleostemin [Oryza sativa (japonicacultivar-group)] 362 77186 0 85 dbj|BAD27898.1|putative PPpentatricopeptide (PPR) repeat- containing protein [Oryza sativa(japonica cultivar-group)] 363 77103 1.00E−174 66ref|NP_568580.1|catalytic LL DS [Arabidopsis thaliana] 364 771392.00E−93 58 ref|NP_171690.1|PFC1 (PALEFACE CS SS 1) [Arabidopsisthaliana] 365 77187 1.00E−160 83 ref|NP_913437.1|3-methyl-2- CS PP LLPEG oxobutanoate hydroxy-methyl- transferase-like protein [Oryza sativa(japonica cultivar-group)] 366 77140 0 77 dbj|BAD36145.1|membraneprotein CK PEG SS PTM1-like [Oryza sativa (japonica cultivar-group)] 36777164 0 91 gb|ABF93778.1|DNA polymerase PP HS delta small subunit,putative, expressed [Oryza sativa (japonica cultivar-group)] 368 77176 076 ref|NP_914476.1|putative LL phytochrome P450 [Oryza sativa (japonicacultivar-group)] 369 77165 1.00E−139 75 ref|NP_914949.1|serine/threonineCK LL LN protein kinase-like protein [Oryza sativa (japonicacultivar-group)] 370 77166 0 85 dbj|BAB75233.1|all3534 [Nostoc sp. CK PPPCC 7120] 371 77155 0 97 emb|CAA35550.1|hycE [Escherichia PP coli] 37277180 2.00E−62 61 gb|ABA99663.1|expressed protein CS PP HS PEG [Oryzasativa (japonica cultivar- group)] 373 77121 1.00E−166 93dbj|BAB05404.1|transcriptional CK repressor of the biotin operon[Bacillus halodurans C-125] 374 77157 0 100ref|NP_531453.1|3,4-dihydroxy-2- CK LL butanone-4-phoshate synthase/GTPcyclohydrolase II [Agrobacterium tumefaciens str. C58] 375 77195 0 94gb|AAM71555.1|mannose-6- PP SS PEG LL phosphate isomerase/mannose-1-phosphate guanylyl transferase [Chlorobium tepidum TLS] 376 771241.00E−180 100 ref|NP_418404.1|biotin--protein PP SS PEG ligase[Escherichia coli K12] 377 77261 0 100 ref|NP_416982.1|hydrogenase 4,PEG subunit [Escherichia coli K12] 378 77273 0 93 dbj|BAB06534.1|glycinePP PEG dehydrogenase subunit 1 [Bacillus halodurans C-125] 379 772031.00E−176 60 dbj|BAD33942.1|putative serine PEG CK carboxypeptidaseprecursor [Oryza sativa (japonica cultivar-group)] 380 77275 4.00E−37 55gb|ABA91490.1|expressed protein SS HS PEG [Oryza sativa (japonicacultivar- group)] 381 77204 2.00E−27 44 ref|XP_469963.1|putativeprotease CK CS inhibitor [Oryza sativa (japonica cultivar-group)] 38277266 7.00E−25 73 gb|AAT93978.1|unknown protein CS PP PEG [Oryza sativa(japonica cultivar- group)] 383 77220 1.00E−169 78dbj|BAD52854.1|putative non- LL phototropic hypocotyl 3 [Oryza sativa(japonica cultivar-group)] 384 77268 4.00E−71 85 gb|ABF95596.1|ETCcomplex I PP SS subunit conserved region family protein, expressed[Oryza sativa (japonica cultivar-group)] 385 77209 9.00E−86 85ref|XP_479456.1|putative 60S PP LL ribosome subunit biogenesis protein[Oryza sativa (japonica cultivar- group)] 386 77269 2.00E−56 70dbj|BAD32031.1|unknown protein SS [Oryza sativa (japonica cultivar-group)] 387 77451 0 71 ref|XP_475231.1|putative PP SSmicrotubule-associated protein [Oryza sativa (japonica cultivar-group)]388 77452 1.00E−106 69 dbj|BAD69045.1|unknown protein PP [Oryza sativa(japonica cultivar- group)] 389 77430 1.00E−160 83dbj|BAD33328.1|putative protein CS PP SS serine/threonine kinase [Oryzasativa (japonica cultivar-group)] 390 77432 0 86ref|NP_849565.1|carbohydrate PP HS PEG transporter/nucleosidetransporter/ sugar porter [Arabidopsis thaliana] gb|AAM19835.1|AT4g35300/F23E12_140 [Arabidopsis thaliana] 391 77433 0 100gb|AAK59487.1|putative cleavage CK PP and polyadenylation specificityfactor [Arabidopsis thaliana] 392 77444 0 96 ref|NP_199947.1|unknownprotein CS PP [Arabidopsis thaliana] 393 77409 0 91ref|NP_001032163.1|unknown protein PP [Arabidopsis thaliana] 394 12313 094 ref|NP_189150.1|QUA1 SP (QUASIMODO1); transferase, transferringglycosyl groups/ transferase, transferring hexosyl groups [Arabidopsisthaliana]

Trait Improvement Screens

DS—Improvement of drought tolerance identified by a soil drought stresstolerance screen: Drought or water deficit conditions impose mainlyosmotic stress on plants. Plants are particularly vulnerable to droughtduring the flowering stage. The drought condition in the screeningprocess disclosed in Example 1B started from the flowering time and wassustained to the end of harvesting. The present invention providesrecombinant DNA that can improve the plant survival rate under suchsustained drought condition. Exemplary recombinant DNA for conferringsuch drought tolerance are identified as such in Table 3. Suchrecombinant DNA may find particular use in generating transgenic plantsthat are tolerant to the drought condition imposed during flowering timeand in other stages of the plant life cycle. As demonstrated from themodel plant screen, in some embodiments of transgenic plants withtrait-improving recombinant DNA grown under such sustained droughtcondition can also have increased total seed weight per plant inaddition to the increased survival rate within a transgenic population,providing a higher yield potential as compared to control plants.

PEG—Improvement of drought tolerance identified by PEG induced osmoticstress tolerance screen: Various drought levels can be artificiallyinduced by using various concentrations of polyethylene glycol (PEG) toproduce different osmotic potentials (Pilon-Smits e.g., (1995) PlantPhysiol. 107:125-130). Several physiological characteristics have beenreported as being reliable indications for selection of plantspossessing drought tolerance. These characteristics include the rate ofseed germination and seedling growth. The traits can be assayedrelatively easily by measuring the growth rate of seedling in PEGsolution. Thus, a PEG-induced osmotic stress tolerance screen is auseful surrogate for drought tolerance screen. As demonstrated from themodel plant screen, embodiments of transgenic plants withtrait-improving recombinant DNA identified in the PEG-induced osmoticstress tolerance screen can survive better drought conditions providinga higher yield potential as compared to control plants.

SS—Improvement of drought tolerance identified by high salinity stresstolerance screen: Three different factors are responsible for saltdamages: (1) osmotic effects, (2) disturbances in the mineralizationprocess, and (3) toxic effects caused by the salt ions, e.g.,inactivation of enzymes. While the first factor of salt stress resultsin the wilting of the plants that is similar to drought effect, theionic aspect of salt stress is clearly distinct from drought. Thepresent invention provides genes that help plants maintain biomass, rootgrowth, and/or plant development in high salinity conditions, which areidentified as such in Table 3. Since osmotic effect is one of the majorcomponents of salt stress, which is common to the drought stress,trait-improving recombinant DNA identified in a high salinity stresstolerance screen can also provide transgenic crops with improved droughttolerance. As demonstrated from the model plant screen, embodiments oftransgenic plants with trait-improving recombinant DNA identified in ahigh salinity stress tolerance screen can survive better droughtconditions and/or high salinity conditions providing a higher yieldpotential as compared to control plants.

HS—Improvement of drought tolerance identified by heat stress tolerancescreen: Heat and drought stress often occur simultaneously, limitingplant growth. Heat stress can cause the reduction in photosynthesisrate, inhibition of leaf growth and osmotic potential in plants. Thus,genes identified by the present invention as heat stress toleranceconferring genes may also impart improved drought tolerance to plants.As demonstrated from the model plant screen, embodiments of transgenicplants with trait-improving recombinant DNA identified in a heat stresstolerance screen can survive better heat stress conditions and/ordrought conditions providing a higher yield potential as compared tocontrol plants.

CK and CS—Improvement of tolerance to cold stress: Low temperature mayimmediately result in mechanical constraints, changes in activities ofmacromolecules, and reduced osmotic potential. In the present invention,two screening conditions, i.e., cold shock tolerance screen (CK) andcold germination tolerance screen (CS), were set up to look fortransgenic plants that display visual growth advantage at lowertemperature. In cold germination tolerance screen, the transgenicArabidopsis plants were exposed to a constant temperature of 8° C. fromplanting until day 28 post plating. The trait-improving recombinant DNAidentified by such screen are particular useful for the production oftransgenic plant that can germinate more robustly in a cold temperatureas compared to the wild type plants. In cold shock tolerance screen, thetransgenic plants were first grown under the normal growth temperatureof 22° C. until day 8 post plating, and subsequently were placed under8° C. until day 28 post plating. As demonstrated from the model plantscreen, embodiments of transgenic plants with trait-improvingrecombinant DNA identified in a cold shock stress tolerance screenand/or a cold germination stress tolerance screen can survive bettercold conditions providing a higher yield potential as compared tocontrol plants.

Improvement of tolerance to multiple stresses: Different kinds ofstresses often lead to identical or similar reaction in the plants.Genes that are activated or inactivated as a reaction to stress caneither act directly in a way the genetic product reduces a specificstress, or they can act indirectly by activating other specific stressgenes. By manipulating the activity of such regulatory genes, i.e.,multiple stress tolerance genes, the plant can be enabled to react todifferent kinds of stresses. For examples, PEP SEQ ID NO: 231 can beused to improve both salt stress tolerance and cold stress tolerance inplants. Of particular interest, plants transformed with PEP SEQ ID NO:233 can resist heat stress, salt stress and cold stress. In addition tothese multiple stress tolerance genes, the stress tolerance conferringgenes provided by the present invention may be used in combinations togenerate transgenic plants that can resist multiple stress conditions.

PP—Improvement of early plant growth and development: It has been knownin the art that to minimize the impact of disease on crop profitability,it is important to start the season with healthy and vigorous plants.This means avoiding seed and seedling diseases, leading to increasednutrient uptake and increased yield potential. Traditionally earlyplanting and applying fertilizer are the methods used for promotingearly seedling vigor. In early development stage, plant embryosestablish only the basic root-shoot axis, a cotyledon storage organ(s),and stem cell populations, called the root and shoot apical meristems,that continuously generate new organs throughout post-embryonicdevelopment. “Early growth and development” used herein encompasses thestages of seed imbibition through the early vegetative phase. Thepresent invention provides genes that are useful to produce transgenicplants that have advantages in one or more processes including, but notlimited to, germination, seedling vigor, root growth and root morphologyunder non-stressed conditions. The transgenic plants starting from amore robust seedling are less susceptible to the fungal and bacterialpathogens that attach germinating seeds and seedling. Furthermore,seedlings with advantage in root growth are more resistant to droughtstress due to extensive and deeper root architecture. Therefore, it canbe recognized by those skilled in the art that genes conferring thegrowth advantage in early stages to plants may also be used to generatetransgenic plants that are more resistant to various stress conditionsdue to improved early plant development. The present invention providessuch exemplary recombinant DNA that confer both the stress tolerance andgrowth advantages to plants, identified as such in Table 3, e.g., PEPSEQ ID NO: 268 which can improve the plant early growth and development,and impart heat and cold tolerance to plants. As demonstrated from themodel plant screen, embodiments of transgenic plants withtrait-improving recombinant DNA identified in the early plantdevelopment screen can grow better under non-stress conditions and/orstress conditions providing a higher yield potential as compared tocontrol plants.

SP—Improvement of late plant growth and development: “Late growth anddevelopment” used herein encompasses the stages of leaf development,flower production, and seed maturity. In certain embodiments, transgenicplants produced using genes that confer growth advantages to plantsprovided by the present invention, identified as such in Table 3,exhibit at least one phenotypic characteristics including, but notlimited to, increased rosette radius, increased rosette dry weight, seeddry weight, silique dry weight, and silique length. On one hand, therosette radius and rosette dry weight are used as the indexes ofphotosynthesis capacity, and thereby plant source strength and yieldpotential of a plant. On the other hand, the seed dry weight, siliquedry weight and silique length are used as the indexes for plant sinkstrength, which are considered as the direct determinants of yield. Asdemonstrated from the model plant screen, embodiments of transgenicplants with trait-improving recombinant DNA identified in the latedevelopment screen can grow better and/or have improved developmentduring leaf development and seed maturation providing a higher yieldpotential as compared to control plants.

LL—Improvement of tolerance to shade stress identified in a low lightscreen: The effects of light on plant development are especiallyprominent at the seedling stage. Under normal light conditions withunobstructed direct light, a plant seeding develops according to acharacteristic photomorphogenic pattern, in which plants have open andexpanded cotyledons and short hypocotyls. Then the plant's energy isdevoted to cotyledon and leaf development while longitudinal extensiongrowth is minimized. Under low light condition where light quality andintensity are reduced by shading, obstruction or high populationdensity, a seedling displays a shade-avoidance pattern, in which theseedling displays a reduced cotyledon expansion, and hypocotylsextension is greatly increased. As the result, a plant under low lightcondition increases significantly its stem length at the expanse ofleaf, seed or fruit and storage organ development, thereby adverselyaffecting of yield. The present invention provides recombinant DNA thatenable plants to have an attenuated shade avoidance response so that thesource of plant can be contributed to reproductive growth efficiently,resulting higher yield as compared to the wild type plants. Asdemonstrated from the model plant screen, embodiments of transgenicplants with trait-improving recombinant DNA identified in a shade stresstolerance screen can have attenuated shade response under shadeconditions providing a higher yield potential as compared to controlplants. The transgenic plants generated by the present invention may besuitable for a higher density planting, thereby resulting increasedyield per unit area.

LN—Improvement of tolerance to low nitrogen availability stress

Nitrogen is a key factor in plant growth and crop yield. The metabolism,growth and development of plants are profoundly affected by theirnitrogen supply. Restricted nitrogen supply alters shoot to root ratio,root development, activity of enzymes of primary metabolism and the rateof senescence (death) of older leaves. All field crops have afundamental dependence on inorganic nitrogenous fertilizer. Sincefertilizer is rapidly depleted from most soil types, it must be suppliedto growing crops two or three times during the growing season. Enhancednitrogen use efficiency by plants should enable crops cultivated underlow nitrogen availability stress condition resulted from low fertilizerinput or poor soil quality.

According to the present invention, transgenic plants generated usingthe recombinant nucleotides, which confer enhanced nitrogen useefficiency, identified as such in Table 3, exhibit one or more desirabletraits including, but not limited to, increased seedling weight, greenerleaves, increased number of rosette leaves, increased or decreased rootlength. One skilled in the art may recognize that the transgenic plantsprovided by the present invention with enhanced nitrogen use efficiencymay also have altered amino acid or protein compositions, increasedyield and/or better seed quality. The transgenic plants of the presentinvention may be productively cultivated under low nitrogen growthconditions, i.e., nitrogen-poor soils and low nitrogen fertilizerinputs, which would cause the growth of wild type plants to cease or tobe so diminished as to make the wild type plants practically useless.The transgenic plants also may be advantageously used to achieve earliermaturing, faster growing, and/or higher yielding crops and/or producemore nutritious foods and animal feedstocks when cultivated usingnitrogen non-limiting growth conditions.

Stacked Traits: The present invention also encompasses transgenic plantswith stacked engineered traits, e.g., a crop having an improvedphenotype resulting from expression of a trait-improving recombinantDNA, in combination with herbicide and/or pest resistance traits. Forexample, genes of the current invention can be stacked with other traitsof agronomic interest, such as a trait providing herbicide resistance,for example a RoundUp Ready® trait, or insect resistance, such as usinga gene from Bacillus thuringensis to provide resistance againstlepidopteran, coliopteran, homopteran, hemiopteran, and other insects.Herbicides for which resistance is useful in a plant include glyphosateherbicides, phosphinothricin herbicides, oxynil herbicides,imidazolinone herbicides, dinitroaniline herbicides, pyridineherbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamideherbicides and gluphosinate herbicides. To illustrate that theproduction of transgenic plants with herbicide resistance is acapability of those of ordinary skill in the art, reference is made toU.S. patent application publications 2003/0106096A1 and 2002/0112260A1and U.S. Pat. Nos. 5,034,322; 5,776,760, 6,107,549 and 6,376,754, all ofwhich are incorporated herein by reference. To illustrate that theproduction of transgenic plants with pest resistance is a capability ofthose of ordinary skill in the art reference is made to U.S. Pat. Nos.5,250,515 and 5,880,275 which disclose plants expressing an endotoxin ofBacillus thuringiensis bacteria, to U.S. Pat. No. 6,506,599 whichdiscloses control of invertebrates which feed on transgenic plants whichexpress dsRNA for suppressing a target gene in the invertebrate, to U.S.Pat. No. 5,986,175 which discloses the control of viral pests bytransgenic plants which express viral replicase, and to U.S. PatentApplication Publication 2003/0150017 A1 which discloses control of pestsby a transgenic plant which express a dsRNA targeted to suppressing agene in the pest, all of which are incorporated herein by reference.

Once one recombinant DNA has been identified as conferring an improvedtrait of interest in transgenic Arabidopsis plants, several methods areavailable for using the sequence of that recombinant DNA and knowledgeabout the protein it encodes to identify homologs of that sequence fromthe same plant or different plant species or other organisms, e.g.,bacteria and yeast. Thus, in one aspect, the invention provides methodsfor identifying a homologous gene with a DNA sequence homologous to anyof SEQ ID NO: 1 through SEQ ID NO: 197, or a homologous protein with anamino acid sequence homologous to any of SEQ ID NO: 198 through SEQ IDNO: 394. In another aspect, the present invention provides the proteinsequences of identified homologs for a sequence listed as SEQ ID NO: 395through SEQ ID NO: 19938. In yet another aspect, the present inventionalso includes linking or associating one or more desired traits, or genefunction with a homolog sequence provided herein.

The trait-improving recombinant DNA and methods of using suchtrait-improving recombinant DNA for generating transgenic plants withimproved traits provided by the present invention are not limited to anyparticular plant species. Indeed, the plants according to the presentinvention may be of any plant species, i.e., may be monocotyledonous ordicotyledonous. Preferably, they will be agricultural useful plants,i.e., plants cultivated by man for purposes of food production ortechnical, particularly industrial applications. Of particular interestin the present invention are corn and soybean plants. The recombinantDNA constructs optimized for soybean transformation and recombinant DNAconstructs optimized for corn transformation are provided by the presentinvention. Other plants of interest in the present invention forproduction of transgenic plants having improved traits include, withoutlimitation, cotton, canola, wheat, sunflower, sorghum, alfalfa, barley,millet, rice, tobacco, fruit and vegetable crops, and turfgrass.

In certain embodiments, the present invention contemplates to use anorthologous gene in generating the transgenic plants with similarlyimproved traits as the transgenic Arabidopsis counterpart. Improvedphysiological properties in transgenic plants of the present inventionmay be confirmed in responses to stress conditions, for example inassays using imposed stress conditions to detect improved responses todrought stress, nitrogen deficiency, cold growing conditions, oralternatively, under naturally present stress conditions, for exampleunder field conditions. Biomass measures may be made on greenhouse orfield grown plants and may include such measurements as plant height,stem diameter, root and shoot dry weights, and, for corn plants, earlength and diameter.

Trait data on morphological changes may be collected by visualobservation during the process of plant regeneration as well as inregenerated plants transferred to soil. Such trait data includescharacteristics such as normal plants, bushy plants, taller plants,thicker stalks, narrow leaves, striped leaves, knotted phenotype,chlorosis, albino, anthocyanin production, or altered tassels, ears orroots. Other enhanced traits may be identified by measurements takenunder field conditions, such as days to pollen shed, days to silking,leaf extension rate, chlorophyll content, leaf temperature, stand,seedling vigor, internode length, plant height, leaf number, leaf area,tillering, brace roots, stay green, stalk lodging, root lodging, planthealth, barreness/prolificacy, green snap, and pest resistance. Inaddition, trait characteristics of harvested grain may be confirmed,including number of kernels per row on the ear, number of rows ofkernels on the ear, kernel abortion, kernel weight, kernel size, kerneldensity and physical grain quality.

To confirm hybrid yield in transgenic corn plants expressing genes ofthe present invention, it may be desirable to test hybrids over multipleyears at multiple locations in a geographical location where maize isconventionally grown, e.g., in Iowa, Illinois or other locations in themidwestern United States, under “normal” field conditions as well asunder stress conditions, e.g., under drought or population densitystress.

Transgenic plants can be used to provide plant parts according to theinvention for regeneration or tissue culture of cells or tissuescontaining the constructs described herein. Plant parts for thesepurposes can include leaves, stems, roots, flowers, tissues, epicotyl,meristems, hypocotyls, cotyledons, pollen, ovaries, cells andprotoplasts, or any other portion of the plant which can be used toregenerate additional transgenic plants, cells, protoplasts or tissueculture. Seeds of transgenic plants are provided by this invention canbe used to propagate more plants containing the trait-improvingrecombinant DNA constructs of this invention. These descendants areintended to be included in the scope of this invention if they contain atrait-improving recombinant DNA construct of this invention, whether ornot these plants are selfed or crossed with different varieties ofplants.

The various aspects of the invention are illustrated by means of thefollowing examples which are in no way intended to limit the full breathand scope of claims.

EXAMPLES Example 1 Identification of Recombinant DNA that ConfersImproved Trait(s) to Plants A. Plant Expression Constructs forArabidopsis Transformation

Each gene of interest was amplified from a genomic or cDNA library usingprimers specific to sequences upstream and downstream of the codingregion. Transformation vectors were prepared to constitutivelytranscribe DNA in either sense orientation (for enhanced proteinexpression) or anti-sense orientation (for endogenous gene suppression)under the control of an enhanced Cauliflower Mosaic Virus 35S promoter(U.S. Pat. No. 5,359,142) directly or indirectly (Moore, e.g., PNAS95:376-381, 1998; Guyer, e.g., Genetics 149: 633-639, 1998;International patent application NO. PCT/EP98/07577). The transformationvectors also contain a bar gene as a selectable marker for resistance toglufosinate herbicide. The transformation of Arabidopsis plants wascarried out using the vacuum infiltration method known in the art(Bethtold, e.g., Methods Mol. Biol. 82:259-66, 1998). Seeds harvestedfrom the plants, named as T1 seeds, were subsequently grown in aglufosinate-containing selective medium to select for plants which wereactually transformed and which produced T2 transgenic seed.

B. Soil Drought Tolerance Screen

This example describes a soil drought tolerance screen to identifyArabidopsis plants transformed with recombinant DNA that wilt lessrapidly and/or produce higher seed yield when grown in soil underdrought conditions

T2 seeds were sown in flats filled with Metro/Mix® 200 (The Scotts®Company, USA). Humidity domes were added to each flat and flats wereassigned locations and placed in climate-controlled growth chambers.Plants were grown under a temperature regime of 22° C. at day and 20° C.at night, with a photoperiod of 16 hours and average light intensity of170 μmol/m²/s. After the first true leaves appeared, humidity domes wereremoved. The plants were sprayed with glufosinate herbicide and put backin the growth chamber for 3 additional days. Flats were watered for 1hour the week following the herbicide treatment. Watering was continuedevery seven days until the flower bud primordia became apparent, atwhich time plants were watered for the last time.

To identify drought tolerant plants, plants were evaluated for wiltingresponse and seed yield. Beginning ten days after the last watering,plants were examined daily until 4 plants/line had wilted. In the nextsix days, plants were monitored for wilting response. Five droughtscores were assigned according to the visual inspection of thephenotypes: 1 for healthy, 2 for dark green, 3 for wilting, 4 severewilting, and 5 for dead. A score of 3 or higher was considered aswilted.

At the end of this assay, seed yield measured as seed weight per plantunder the drought condition was characterized for the transgenic plantsand their controls and analyzed as a quantitative response according toexample 1M.

Two approaches were used for statistical analysis on the wiltingresponse. First, the risk score was analyzed for wilting phenotype andtreated as a qualitative response according to the example 1L.Alternatively, the survival analysis was carried out in which theproportions of wilted and non-wilted transgenic and control plants werecompared over each of the six days under scoring and an overall log ranktest was performed to compare the two survival curves using S-PLUSstatistical software (S-PLUS 6, Guide to statistics, Insightful,Seattle, Wash., USA). A list of recombinant DNA constructs which improvedrought tolerance in transgenic plants is illustrated in Table 4.

TABLE 4 Time to wilting PEP Drought score Seed yield Risk SEQ ConstructNomination Delta P- Delta P- score P- ID NO ID ID Orientation mean valuemean value mean value 198 11029 CGPG106 SENSE 0.109 0.381 0.072 0.7170.119 1.000 292 73136 CGPG5764 SENSE 0.030 0.090 0.136 0.067 0.104 1.000284 73165 CGPG5661 SENSE −0.031 0.504 0.526 0.018 −0.075 1.000 260 73628CGPG5025 SENSE 0.342 0.038 −0.474 0.022 0.208 1.000 291 73981 CGPG5757SENSE 0.517 0.026 −0.096 0.000 0.573 1.000 207 74065 CGPG1828 ANTI-−0.078 0.164 0.646 0.027 0.048 1.000 SENSE 263 74217 CGPG5144 SENSE−0.026 0.414 0.418 0.032 0.071 1.000 261 76105 CGPG5041 SENSE 0.2880.029 −1.780 0.027 0.173 1.000 328 76576 CGPG7281 SENSE 0.241 0.032−0.555 0.442 0.212 1.000 327 77536 CGPG7272 SENSE 0.089 0.194 1.0590.001 0.042 1.000 226 19767 CGPG3918 SENSE 0.117 0.038 0.164 0.296 / /237 19947 CGPG4069 SENSE −0.009 0.852 −0.104 0.504 / / 242 70994CGPG4122 SENSE 0.040 0.021 −0.131 0.447 / / 363 77103 CGPG9134 SENSE0.145 0.023 −0.499 0.096 / /

If p<0.05 and delta or risk score mean >0, the transgenic plants showedstatistically significant trait improvement as compared to the reference(p value, of the delta of a quantitative response or of the risk scoreof a qualitative response, is the probability that the observeddifference between the transgenic plants and the reference occur bychance) If p<0.2 and delta or risk score mean >0, the transgenic plantsshowed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as setforth in SEQ ID NO: 226, 237, 242, or 363 showed improved droughttolerance evidenced by the second criteria as illustrated in Example 1Land 1M.

C. Heat Stress Tolerance Screen

Under high temperatures, Arabidopsis seedlings become chlorotic and rootgrowth is inhibited. This example sets forth the heat stress tolerancescreen to identify Arabidopsis plants transformed with the gene ofinterest that are more resistant to heat stress based on primarily theirseedling weight and root growth under high temperature.

T2 seeds were plated on ½× MS salts, 1/% phytagel, with 10 μg/ml BASTA(7 per plate with 2 control seeds; 9 seeds total per plate). Plates wereplaced at 4° C. for 3 days to stratify seeds. Plates were then incubatedat room temperature for 3 hours and then held vertically for 11additional days at temperature of 34° C. at day and 20° C. at night.Photoperiod was 16 h. Average light intensity was ˜140 μmol/m²/s. After14 days of growth, plants were scored for glufosinate resistance, rootlength, final growth stage, visual color, and seedling fresh weight. Aphotograph of the whole plate was taken on day 14.

The seedling weight and root length were analyzed as quantitativeresponses according to example 1M. The final grow stage at day 14 wasscored as success if 50% of the plants had reached 3 rosette leaves andsize of leaves are greater than 1 mm (Boyes, e.g., (2001) The Plant Cell13, 1499-1510). The growth stage data was analyzed as a qualitativeresponse according to example 1L. A list of recombinant DNA constructsthat improve heat tolerance in transgenic plants illustrated in Table 5.

TABLE 5 Growth stage Root length at day 14 Seedling weight PEP at day 14Risk at day 14 SEQ Construct Nomination Delta P- score P- Delta P- ID IDID Orientation mean value mean value mean value 239 70985 CGPG4088 SENSE0.480 0.036 1.176 0.196 1.333 0.018 233 70987 CGPG4048 SENSE 0.228 0.021−0.036 0.670 1.214 0.002 268 72001 CGPG5221 SENSE 0.273 0.004 0.2100.330 1.393 0.009 274 72753 CGPG5540 SENSE 0.434 0.006 0.414 0.258 1.1000.006 276 73954 CGPG5577 SENSE 0.406 0.002 0.455 0.282 1.274 0.000 32576178 CGPG7225 SENSE 0.714 0.001 1.093 0.145 1.537 0.000 361 77150CGPG9130 SENSE 0.219 0.032 0.178 0.237 1.159 0.002 367 77164 CGPG9147SENSE 0.317 0.026 0.651 0.099 1.152 0.002 390 77432 CGPG9335 SENSE 0.5810.001 0.457 0.124 1.551 0.000 351 77587 CGPG8107 SENSE 0.488 0.006 0.6280.072 1.328 0.001 348 77928 CGPG8082 SENSE 0.462 0.032 0.191 0.301 1.4980.004 212 16610 CGPG2499 SENSE 0.055 0.640 0.109 0.609 0.983 0.020 22018256 CGPG3363 SENSE 0.070 0.138 0.071 0.541 1.021 0.004 222 19193CGPG3375 SENSE 0.255 0.094 0.267 0.368 1.023 0.003 252 70684 CGPG4588SENSE 0.010 0.781 0.003 0.979 1.002 0.045 230 70948 CGPG3990 SENSE 0.2820.095 0.351 0.335 1.045 0.023 243 70956 CGPG4140 SENSE 0.155 0.313 0.0910.320 1.129 0.018 235 70962 CGPG4057 SENSE 0.469 0.085 0.267 0.508 1.4100.007 241 70963 CGPG4121 SENSE −0.002 0.979 −0.131 0.311 0.976 0.005 24270994 CGPG4122 SENSE 0.140 0.574 1.204 0.096 1.207 0.030 244 70995CGPG4154 SENSE 0.374 0.112 1.640 0.171 1.328 0.013 208 72783 CGPG2206SENSE 0.387 0.057 0.053 0.509 1.013 0.019 255 72807 CGPG4912 SENSE 0.3570.071 0.443 0.020 1.099 0.009 259 72825 CGPG5001 SENSE 0.171 0.349 0.2130.234 1.118 0.012 287 73120 CGPG5704 SENSE 0.096 0.490 0.193 0.364 1.0800.033 280 73127 CGPG5640 SENSE −0.220 0.077 −0.194 0.030 0.643 0.014 29373172 CGPG5783 SENSE 0.283 0.197 0.887 0.162 1.154 0.007 310 73411CGPG6440 SENSE 0.198 0.389 0.631 0.345 1.320 0.003 260 73628 CGPG5025SENSE 0.406 0.149 1.475 0.365 1.169 0.016 316 74566 CGPG6796 SENSE 0.6270.092 0.777 0.466 1.714 0.014 338 75654 CGPG7804 SENSE 0.292 0.237 0.8040.315 0.940 0.017 340 75657 CGPG7828 SENSE 0.390 0.067 1.222 0.307 1.5210.011 213 76602 CGPG2653 SENSE 0.036 0.748 0.092 0.670 0.752 0.025 37277180 CGPG9180 SENSE 0.068 0.555 −0.045 0.177 0.871 0.022 380 77275CGPG9236 SENSE 0.282 0.125 0.971 0.285 1.027 0.013 297 77312 CGPG5927SENSE 0.201 0.344 0.710 0.372 1.039 0.032 320 77518 CGPG6953 SENSE 0.2380.344 0.069 0.655 1.188 0.015 342 77549 CGPG7933 SENSE 0.184 0.180−0.012 / 0.978 0.007 347 77580 CGPG8062 SENSE 0.157 0.143 −0.074 / 0.8910.049 317 77610 CGPG6805 SENSE 0.023 0.937 −0.063 / 1.207 0.029 31877618 CGPG6810 SENSE 0.002 0.942 −0.074 / 1.015 0.006 353 77619 CGPG8152SENSE 0.213 0.316 −0.076 0.457 0.956 0.019 355 77629 CGPG8377 SENSE0.095 0.124 −0.063 / 1.133 0.005 334 77816 CGPG7529 SENSE 0.033 0.7050.856 0.385 0.611 0.038

If p<0.05 and delta or risk score mean >0, the transgenic plants showedstatistically significant trait improvement as compared to thereference. If p<0.2 and delta or risk score mean >0, the transgenicplants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as setforth in SEQ ID NO: 237, 307, 313, 327, 330, 349, 366, or 387 showedimproved heat stress tolerance evidenced by the second criteria asillustrated in Example 1L and 1M.

D. Salt Stress Tolerance Screen

This example sets forth the high salinity stress screen to identifyArabidopsis plants transformed with the gene of interest that aretolerant to high levels of salt based on their rate of development, rootgrowth and chlorophyll accumulation under high salt conditions.

T2 seeds were plated on glufosinate selection plates containing 90 mMNaCl and grown under standard light and temperature conditions. Allseedlings used in the experiment were grown at a temperature of 22° C.at day and 20° C. at night, a 16-hour photoperiod, an average lightintensity of approximately 120 umol/m². On day 11, plants were measuredfor primary root length. After 3 more days of growth (day 14), plantswere scored for transgenic status, primary root length, growth stage,visual color, and the seedlings were pooled for fresh weightmeasurement. A photograph of the whole plate was also taken on day 14.

The seedling weight and root length were analyzed as quantitativeresponses according to example 1M. The final growth stage at day 14 wasscored as success if 50% of the plants reached 3 rosette leaves and sizeof leaves are greater than 1 mm (Boyes, D.C., et al., (2001), The PlantCell 13, 1499/1510). The growth stage data was analyzed as a qualitativeresponse according to example 1L. A list of recombinant DNA constructsthat improve high salinity tolerance in transgenic plants illustrated inTable 6.

TABLE 6 Root length Root length Growth stage Seedling weight PEP at day11 at day 14 at day 14 at day 14 SEQ Construct Delta P- Delta P- DeltaP- Delta P- ID ID Orientation mean value mean value mean value meanvalue 220 18256 SENSE 0.192 0.232 0.236 0.080 1.291 0.151 0.598 0.039214 18456 SENSE 0.213 0.089 0.209 0.197 2.062 0.197 0.476 0.046 23219973 SENSE 0.289 0.012 0.299 0.002 0.976 0.054 1.002 0.013 231 70201SENSE 0.363 0.030 0.403 0.052 2.083 0.229 1.057 0.014 243 70956 SENSE0.006 0.923 0.128 0.016 −0.045 NA −0.364 0.371 238 70969 SENSE 0.4350.091 0.471 0.027 0.653 0.473 0.994 0.058 240 70971 SENSE 0.337 0.0630.257 0.041 1.418 0.226 0.593 0.030 233 70987 SENSE 0.363 0.030 0.2840.044 1.434 0.380 0.865 0.012 273 72788 SENSE 0.484 0.019 0.456 0.0161.813 0.314 0.929 0.008 256 72811 SENSE 0.385 0.024 0.409 0.002 2.9680.031 0.805 0.004 287 73120 SENSE 0.341 0.001 0.244 0.019 0.613 0.1030.755 0.015 280 73127 SENSE 0.501 0.028 0.395 0.055 0.212 0.175 0.7390.023 289 73134 SENSE 0.226 0.134 0.266 0.045 0.152 0.487 0.378 0.056257 73235 SENSE 0.431 0.020 0.360 0.018 1.618 0.327 0.650 0.009 24573306 SENSE 0.228 0.046 0.219 0.018 0.249 0.217 0.708 0.008 253 73330SENSE 0.196 0.225 0.186 0.029 0.000 NA 0.543 0.045 316 74566 SENSE 0.3310.001 0.076 0.422 0.597 0.031 0.716 0.003 312 74688 SENSE 0.480 0.0760.415 0.031 0.000 NA 0.808 0.062 376 77124 SENSE 0.340 0.034 0.322 0.0152.306 0.137 0.411 0.168 364 77139 SENSE 0.339 0.096 0.287 0.041 1.6830.294 0.602 0.039 375 77195 SENSE 0.445 0.009 0.345 0.010 0.367 0.1850.582 0.022 384 77268 SENSE 0.495 0.047 0.468 0.102 1.906 0.079 0.9920.042 386 77269 SENSE 0.454 0.038 0.426 0.004 1.996 0.065 1.136 0.001380 77275 SENSE 0.457 0.008 0.487 0.040 2.119 0.154 1.070 0.010 29777312 SENSE 0.335 0.086 0.361 0.013 0.960 0.208 0.723 0.063 389 77430SENSE 0.303 0.030 0.360 0.004 0.131 0.495 0.894 0.022 320 77518 SENSE0.171 0.017 0.185 0.026 −0.134 0.443 0.339 0.109 345 77570 SENSE 0.3260.091 0.241 0.134 1.396 0.090 0.862 0.026 351 77587 SENSE 0.178 0.0830.096 0.665 0.995 0.426 0.632 0.036 353 77619 SENSE 0.389 0.058 0.3980.011 1.913 0.257 0.857 0.040 355 77629 SENSE 0.404 0.043 0.389 0.0192.523 0.083 0.826 0.040 336 77821 SENSE 0.393 0.063 0.444 0.033 0.1960.228 0.619 0.015 356 77832 SENSE 0.134 0.238 0.304 0.011 0.914 0.1560.413 0.050 352 77933 SENSE 0.112 0.044 0.165 0.009 0.749 0.482 0.5170.171 347 77580 SENSE −0.019 0.814 0.087 0.018 −0.158 0.151 0.124 0.204

If p<0.05 and delta or risk score mean >0, the transgenic plants showedstatistically significant trait improvement as compared to thereference. If p<0.2 and delta or risk score mean >0, the transgenicplants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as setforth in SEQ ID NO: 237, 242, 255, 307, 313, 327, 330, 349, 366, or 387showed improved salt stress tolerance evidenced by the second criteriaas illustrated in Example 1L.

E. Polyethylene Glycol (PEG) Induced Osmotic Stress Tolerance Screen

There are numerous factors, which can influence seed germination andsubsequent seedling growth, one being the availability of water. Genes,which can directly affect the to success rate of germination and earlyseedling growth, are potentially useful agronomic traits for improvingthe germination and growth of crop plants under drought stress. In thisassay, PEG was used to induce osmotic stress on germinating transgeniclines of Arabidopsis thaliana seeds in order to screen for osmoticallyresistant seed lines.

T2 seeds were plated on BASTA selection plates containing 3% PEG andgrown under standard light and temperature conditions. Seeds were platedon each plate containing 3% PEG, ½× MS salts, 1% phytagel, and 10 μg/mlglufosinate. Plates were placed at 4° C. for 3 days to stratify seeds.On day 11, plants were measured for primary root length. After 3 moredays of growth, i.e., at day 14, plants were scored for transgenicstatus, primary root length, growth stage, visual color, and theseedlings were pooled for fresh weight measurement. A photograph of thewhole plate was taken on day 14.

Seedling weight and root length were analyzed as quantitative responsesaccording to example 1M. The final growth stage at day 14 was scored assuccess or failure based on whether the plants reached 3 rosette leavesand size of leaves are greater than 1 mm. The growth stage data wasanalyzed as a qualitative response according to example 1L. A list ofrecombinant DNA constructs that improve osmotic stress tolerance intransgenic plants illustrated in Table 7.

TABLE 7 Root length Root length Growth stage Seedling weight PEP at day11 at day 14 at day 14 at day 14 SEQ Construct Delta P- Delta P- DeltaP- Delta P- ID ID Orientation mean value mean value mean value meanvalue 199 12223 SENSE / / / / / / 0.353 0.024 214 18456 SENSE 0.3200.018 0.274 0.033 3.109 0.073 0.553 0.006 302 70812 SENSE 0.195 0.0780.078 0.296 4 0.000 0.363 0.016 234 70950 SENSE 0.322 0.056 0.427 0.0164 0.000 0.381 0.127 239 70985 SENSE 0.270 0.066 0.221 0.005 4 0.0000.372 0.007 233 70987 SENSE 0.227 0.205 0.128 0.470 4 0.000 0.495 0.000228 70992 SENSE 0.369 0.002 0.370 0.013 4 0.000 0.574 0.027 244 70995SENSE 0.379 0.049 0.320 0.065 4 0.000 0.491 0.003 208 72783 SENSE 0.1540.084 0.043 0.553 4 0.000 0.264 0.044 255 72807 SENSE 0.165 0.080 0.1580.150 4 0.000 0.333 0.047 256 72811 SENSE 0.489 0.052 0.491 0.050 40.000 0.784 0.016 259 72825 SENSE 0.423 0.055 0.388 0.080 2.741 0.1610.608 0.020 295 72946 SENSE 0.345 0.053 0.473 0.050 0.898 0.632 0.4150.181 286 73026 SENSE 0.636 0.021 0.619 0.028 2.741 0.161 0.752 0.045290 73123 SENSE 0.460 0.048 0.445 0.007 NA NA 0.609 0.069 278 73161SENSE 0.510 0.024 0.506 0.006 2.624 0.246 0.626 0.073 284 73165 SENSE0.304 0.101 0.285 0.044 2.499 0.270 0.643 0.077 293 73172 SENSE 0.1970.056 0.137 0.204 2.374 0.293 0.660 0.081 262 73242 SENSE 0.305 0.0290.332 0.022 2.249 0.317 0.677 0.085 265 73256 SENSE 0.325 0.084 0.1820.178 2.124 0.341 0.694 0.089 308 73485 SENSE 0.133 0.070 0.039 0.5831.999 0.365 0.711 0.093 291 73981 SENSE 0.239 0.147 0.238 0.281 1.8740.389 0.728 0.096 316 74566 SENSE 0.451 0.004 0.248 0.118 1.749 0.4120.745 0.100 312 74688 SENSE 0.190 0.191 0.102 0.520 1.624 0.436 0.7620.104 330 74863 SENSE 0.174 0.164 0.216 0.109 1.499 0.460 0.779 0.108366 77140 SENSE 0.644 0.005 0.572 0.015 1.374 0.484 0.796 0.112 36177150 SENSE 0.313 0.047 0.359 0.044 1.249 0.508 0.813 0.116 365 77187SENSE 0.273 0.084 0.032 0.763 1.124 0.532 0.830 0.120 375 77195 SENSE0.319 0.021 0.256 0.041 NA NA 0.847 0.124 379 77203 SENSE 0.113 0.4850.019 0.829 0.999 0.555 0.864 0.127 377 77261 SENSE 0.115 0.230 0.0850.315 0.874 0.579 0.881 0.131 270 77308 SENSE 0.174 0.321 0.074 0.7000.749 0.603 0.898 0.135 346 77338 SENSE 0.227 0.158 0.196 0.272 0.6240.627 0.915 0.139 350 77357 SENSE 0.169 0.300 0.259 0.066 2.715 0.1690.303 0.020 390 77432 SENSE 0.306 0.043 0.122 0.270 2.57 0.214 0.7340.019 347 77580 SENSE 0.300 0.107 0.240 0.009 2.288 0.119 0.500 0.179353 77619 SENSE 0.142 0.216 0.139 0.002 4 0.000 0.129 0.582 356 77832SENSE 0.236 0.006 0.266 0.001 1.333 0.435 0.286 0.037 343 77917 SENSE0.174 0.163 0.160 0.006 4 0.000 0.330 0.009

If p<0.05 and delta or risk score mean >0, the transgenic plants showedstatistically significant trait improvement as compared to thereference.

If p<0.2 and delta or risk score mean >0, the transgenic plants showed atrend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as setforth in SEQ ID NO: 209, 245, 260, 274, 282, 310, 321, 325, 336, 372,376, 378, 380, or 382 showed improved osmotic stress tolerance evidencedby the second criteria as illustrated in Example 1L and 1M.

F. Cold Shock Tolerance Screen

This example set forth a screen to identify Arabidopsis plantstransformed with the genes of interest that are more tolerant to coldstress subjected during day 8 to day 28 after seed planting. Duringthese crucial early stages, seedling growth and leaf area increase weremeasured to assess tolerance when Arabidopsis seedlings were exposed tolow temperatures. Using this screen, genetic alterations can be foundthat enable plants to germinate and grow better than wild type plantsunder sudden exposure to low temperatures.

Eleven seedlings from T2 seeds of each transgenic line plus one controlline were plated together on a plate containing ½× Gamborg Salts with0.8 Phytagel™, 1% Phytagel, and 0.3% Sucrose. Plates were then orientedhorizontally and stratified for three days at 4° C. At day three, plateswere removed from stratification and exposed to standard conditions (16hr photoperiod, 22° C. at day and 20° C. at night) until day 8. At dayeight, plates were removed from standard conditions and exposed to coldshock conditions (24 hr photoperiod, 8° C. at both day and night) untilthe final day of the assay, i.e., day 28. Rosette areas were measured atday 8 and day 28, which were analyzed as quantitative responsesaccording to example 1M. A list of recombinant nucleotides that improvecold shock stress tolerance in plants illustrated in Table 8.

TABLE 8 Rosette area Rosette area at day 28 Rosette area PEP at day 8Risk difference SEQ Construct Nomination Delta P- score P- Delta P- IDID ID Orientation mean value mean value mean value 199 12223 CGPG1133SENSE 0.112 0.617 0.334 0.014 0.273 0.143 218 18231 CGPG3274 SENSE−0.131 0.269 1.144 0.002 1.179 0.003 215 18414 CGPG3002 SENSE 0.9970.034 1.665 0.001 1.895 0.001 214 18456 CGPG2813 SENSE 0.099 0.757 0.7990.001 0.684 0.001 233 70987 CGPG4048 SENSE 0.864 0.136 1.213 0.010 1.1460.002 244 70995 CGPG4154 SENSE 1.088 0.008 1.262 0.023 1.407 0.025 20872783 CGPG2206 SENSE 0.814 0.023 0.957 0.018 0.899 0.052 273 72788CGPG5535 SENSE 0.262 0.058 1.090 0.016 1.212 0.021 256 72811 CGPG4926SENSE 0.880 0.058 0.669 0.002 0.715 0.018 259 72825 CGPG5001 SENSE 0.0320.887 0.338 0.071 0.357 0.027 281 73033 CGPG5646 SENSE 0.322 0.394 1.3110.012 1.480 0.017 293 73172 CGPG5783 SENSE 0.395 0.209 1.065 0.030 1.0990.074 253 73330 CGPG4765 SENSE 0.347 0.515 0.835 0.065 1.043 0.007 26473735 CGPG5171 SENSE 0.624 0.076 0.655 0.001 0.718 0.005 315 74460CGPG6747 SENSE 0.452 0.005 0.542 0.043 0.455 0.154 296 74746 CGPG5856SENSE 0.304 0.346 0.978 0.001 1.061 0.007 299 75240 CGPG5957 SENSE 0.4790.153 0.652 0.143 0.875 0.039 338 75654 CGPG7804 SENSE 0.531 0.140 1.7120.010 1.979 0.015 339 75692 CGPG7823 SENSE 0.526 0.063 2.027 0.001 2.2460.001 323 76161 CGPG7168 SENSE 0.768 0.000 1.810 0.004 2.146 0.003 21776532 CGPG3235 SENSE 0.921 0.008 1.216 0.001 1.324 0.002 357 76802CGPG8987 SENSE 0.468 0.097 1.578 0.000 1.872 0.000 358 76829 CGPG9013SENSE −0.434 0.329 0.709 0.010 0.314 0.118 373 77121 CGPG9183 SENSE0.081 0.385 0.757 0.023 0.698 0.014 366 77140 CGPG9145 SENSE −0.2210.165 0.896 0.007 1.043 0.005 374 77157 CGPG9186 SENSE 0.190 0.566 1.1100.016 1.176 0.015 369 77165 CGPG9155 SENSE 1.573 0.043 0.683 0.038 0.6990.022 370 77166 CGPG9163 SENSE 0.777 0.011 1.432 0.016 1.570 0.025 38177204 CGPG9238 SENSE 0.545 0.139 1.313 0.003 1.436 0.006 391 77433CGPG9341 SENSE 1.054 0.103 0.895 0.027 0.659 0.084 345 77570 CGPG8015SENSE 0.538 0.215 0.866 0.025 0.898 0.023 353 77619 CGPG8152 SENSE−0.589 0.322 0.503 0.068 0.558 0.049 354 77621 CGPG8166 SENSE 0.0530.863 0.716 0.044 0.792 0.040 203 13411 CGPG1301 SENSE 0.183 0.427 0.5770.033 0.573 0.031 209 17210 CGPG2217 SENSE −0.244 0.685 0.162 0.0380.156 0.104 220 18256 CGPG3363 SENSE 0.272 0.400 0.639 0.008 0.719 0.020289 73134 CGPG5721 SENSE 0.014 0.947 1.053 0.000 1.062 0.025 263 74217CGPG5144 SENSE 0.168 0.390 0.261 0.043 0.206 0.075 379 77203 CGPG9230SENSE 0.606 0.051 0.620 0.032 0.778 0.039

If p<0.05 and delta or risk score mean >0, the transgenic plants showedstatistically significant trait improvement as compared to the reference(p value, of the delta of a quantitative response or of the risk scoreof a qualitative response, is the probability that the observeddifference between the transgenic plants and the reference occur bychance) If p<0.2 and delta or risk score mean >0, the transgenic plantsshowed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as setforth in SEQ ID NO: 255, 272, or 330 showed improved cold stresstolerance evidenced by the second criterial as illustrated in Example1L.

G. Cold Germination Tolerance Screen

This example sets forth a screen to identify Arabidopsis plantstransformed with the genes of interests are resistant to cold stressbased on their rate of development, root growth and chlorophyllaccumulation under low temperature conditions.

T2 seeds were plated and all seedlings used in the experiment were grownat 8° C. Seeds were first surface disinfested using chlorine gas andthen seeded on assay plates containing an aqueous solution of ½×Gamborg's B/5 Basal Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo.,USA G/5788), 1% Phytagel™ (Sigma-Aldrich, P-8169), and 10 ug/mlglufosinate with the final pH adjusted to 5.8 using KOH. Test plateswere held vertically for 28 days at a constant temperature of 8° C., aphotoperiod of 16 hr, and average light intensity of approximately 100umol/m²/s. At 28 days post plating, root length was measured, growthstage was observed, the visual color was assessed, and a whole platephotograph was taken.

The root length at day 28 was analyzed as a quantitative responseaccording to example 1M. The growth stage at day 7 was analyzed as aqualitative response according to example 1L. A list of recombinant DNAconstructs that improve cold stress tolerance in transgenic plantsillustrated in Table 9.

TABLE 9 Root length Growth stage PEP at day 28 at day 28 SEQ ConstructNomination Delta P- Delta P- ID ID ID Orientation mean value mean value209 17210 CGPG2217 SENSE 0.164 0.089 4.000 0.000 219 18232 CGPG3275SENSE −0.081 0.613 4.000 0.000 226 19767 CGPG3918 SENSE 0.212 0.0074.000 0.000 227 19774 CGPG3920 SENSE 0.140 0.011 4.000 0.000 237 19947CGPG4069 SENSE / / 4.000 0.000 231 70201 CGPG3994 SENSE 0.311 0.0104.000 0.000 252 70684 CGPG4588 SENSE 0.335 0.209 4.000 0.000 236 70915CGPG4058 SENSE 0.231 0.258 4.000 0.000 243 70956 CGPG4140 SENSE 0.1130.525 4.000 0.000 241 70963 CGPG4121 SENSE 0.210 0.252 4.000 0.000 23970985 CGPG4088 SENSE 0.283 0.063 4.000 0.000 233 70987 CGPG4048 SENSE0.392 0.202 4.000 0.000 228 70992 CGPG3962 SENSE 0.008 0.959 4.000 0.000244 70995 CGPG4154 SENSE 0.426 0.002 4.000 0.000 268 72001 CGPG5221SENSE 0.070 0.728 4.000 0.000 274 72753 CGPG5540 SENSE 0.189 0.010 2.8890.121 273 72788 CGPG5535 SENSE 0.548 0.009 4.000 0.000 287 73120CGPG5704 SENSE 0.401 0.146 4.000 0.000 290 73123 CGPG5728 SENSE 0.1710.166 4.000 0.000 277 73137 CGPG5587 SENSE 0.382 0.019 4.000 0.000 28373141 CGPG5659 SENSE 0.348 0.029 1.891 0.219 285 73155 CGPG5684 SENSE0.194 0.105 4.000 0.000 278 73161 CGPG5594 SENSE 0.327 0.018 2.889 0.121265 73256 CGPG5194 SENSE 0.176 0.473 4.000 0.000 266 73260 CGPG5200SENSE 0.256 0.047 4.000 0.000 253 73330 CGPG4765 SENSE 0.229 0.016 4.0000.000 310 73411 CGPG6440 SENSE 0.232 0.213 4.000 0.000 260 73628CGPG5025 SENSE 0.304 0.040 1.330 0.467 204 73944 CGPG1458 SENSE 0.4260.026 4.000 0.000 207 74065 CGPG1828 ANTI- 0.215 0.026 2.599 0.205 SENSE248 74536 CGPG442 SENSE −0.054 0.431 4.000 0.000 312 74688 CGPG6653SENSE 0.645 0.006 4.000 0.000 340 75657 CGPG7828 SENSE 0.394 0.017 2.6800.180 325 76178 CGPG7225 SENSE 0.334 0.107 4.000 0.000 321 76460CGPG7121 SENSE 0.136 0.381 4.000 0.000 305 76527 CGPG6306 SENSE 0.2380.001 0.335 0.421 217 76532 CGPG3235 SENSE 0.250 0.331 4.000 0.000 21376602 CGPG2653 SENSE 0.283 0.387 4.000 0.000 306 77020 CGPG6318 SENSE0.507 0.038 0.000 0.000 364 77139 CGPG9137 SENSE 0.445 0.005 0.000 0.000372 77180 CGPG9180 SENSE 0.370 0.046 4.000 0.000 365 77187 CGPG9141SENSE 0.456 0.036 4.000 0.000 381 77204 CGPG9238 SENSE 0.314 0.002 4.0000.000 382 77266 CGPG9259 SENSE 0.017 0.948 4.000 0.000 389 77430CGPG9322 SENSE 0.095 0.233 4.000 0.000 392 77444 CGPG9344 SENSE 0.1710.292 4.000 0.000 319 77517 CGPG6952 SENSE 0.132 0.001 2.507 0.235 32777536 CGPG7272 SENSE −0.084 0.675 4.000 0.000 351 77587 CGPG8107 SENSE0.415 0.112 4.000 0.000 317 77610 CGPG6805 SENSE 0.267 0.180 4.000 0.000356 77832 CGPG8976 SENSE 0.153 0.579 4.000 0.000

If p<0.05 and delta or risk score mean >0, the transgenic plants showedstatistically significant trait improvement as compared to thereference. If p<0.2 and delta or risk score mean >0, the transgenicplants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as setforth in SEQ ID NO: 347 showed improved cold stress tolerance evidencedby the second criteria as illustrated in Example 1L and 1M.

H. Shade Tolerance Screen

Plants undergo a characteristic morphological response in shade thatincludes the elongation of the petiole, a change in the leaf angle, anda reduction in chlorophyll content. While these changes may confer acompetitive advantage to individuals, in a monoculture the shadeavoidance response is thought to reduce the overall biomass of thepopulation. Thus, genetic alterations that prevent the shade avoidanceresponse may be associated with higher yields. Genes that favor growthunder low light conditions may also promote yield, as inadequate lightlevels frequently limit yield. This protocol describes a screen to lookfor Arabidopsis plants that show an attenuated shade avoidance responseand/or grow better than control plants under low light intensity. Ofparticular interest, we were looking for plants that didn't extend theirpetiole length, had an increase in seedling weight relative to thereference and had leaves that were more close to parallel with the platesurface.

T2 seeds were plated on glufosinate selection plates with ½ MS medium.Seeds were sown on ½× MS salts, 1% Phytagel, 10 ug/ml BASTA. Plants weregrown on vertical plates at a temperature of 22° C. at day, 20° C. atnight and under low light (approximately 30 uE/m²/s, far/red ratio(655/665/725/735) ˜0.35 using PLAQ lights with GAM color filter #680).Twenty-three days after seedlings were sown, measurements were recordedincluding seedling status, number of rosette leaves, status of flowerbud, petiole leaf angle, petiole length, and pooled fresh weights. Adigital image of the whole plate was taken on the measurement day.Seedling weight and petiole length were analyzed as quantitativeresponses according to example 1M. The number of rosette leaves,flowering bud formation and leaf angel were analyzed as qualitativeresponses according to example 1L.

A list of recombinant DNA constructs that improve shade tolerance inplants illustrated in Table 10.

TABLE 10 Leaf angle Seedling weight Petiole length PEP at day 23 at day23 at day 23 SEQ Construct Nomination RS P- Delta P- Delta P- ID ID IDOrientation mean value mean value mean value 251 70755 CGPG4473 SENSE NANA −0.751 0.017 −0.307 0.059 244 70995 CGPG4154 SENSE NA NA 0.251 0.0700.008 0.944 275 72709 CGPG5568 SENSE NA NA −0.903 0.070 −0.608 0.019 21072724 CGPG2292 SENSE NA NA −0.707 0.098 −0.638 0.020 272 72774 CGPG5518SENSE NA NA −0.268 0.110 −0.195 0.087 258 72813 CGPG4977 SENSE NA NA−0.469 0.092 −0.186 0.054 294 73020 CGPG5791 SENSE NA NA −1.443 0.010−1.265 0.010 288 73133 CGPG5714 SENSE NA NA −0.852 0.009 −0.896 0.012260 73628 CGPG5025 SENSE NA NA 0.311 0.011 0.317 0.146 271 73766CGPG5432 SENSE NA NA −0.988 0.048 −0.671 0.088 314 74435 CGPG6737 SENSENA NA −0.735 0.042 −0.755 0.049 331 74858 CGPG7371 SENSE NA NA −0.6670.010 −0.808 0.008 330 74863 CGPG7316 SENSE NA NA 0.176 0.060 0.1680.515 332 74933 CGPG7457 SENSE NA NA −0.100 0.801 −0.533 0.094 340 75657CGPG7828 SENSE NA NA 0.193 0.059 0.244 0.085 337 75685 CGPG7767 SENSE NANA −0.917 0.075 −0.974 0.049 267 75822 CGPG5210 SENSE NA NA −0.267 0.194−0.297 0.090 324 76171 CGPG7206 SENSE NA NA −0.350 0.065 −0.570 0.049254 76202 CGPG4788 SENSE NA NA −1.153 0.009 −1.713 0.019 326 76467CGPG7267 SENSE NA NA −0.484 0.177 −0.743 0.088 359 76961 CGPG9080 SENSENA NA −0.425 0.098 −0.265 0.051 360 76973 CGPG9081 SENSE NA NA 0.3330.052 0.248 0.003 363 77103 CGPG9134 SENSE NA NA 0.363 0.093 0.198 0.072374 77157 CGPG9186 SENSE NA NA 0.287 0.044 0.107 0.086 369 77165CGPG9155 SENSE NA NA −0.081 0.413 −0.572 0.012 368 77176 CGPG9148 SENSENA NA −0.577 0.025 −0.651 0.085 365 77187 CGPG9141 SENSE NA NA 0.3350.002 0.263 0.046 385 77209 CGPG9278 SENSE NA NA 0.671 0.027 0.451 0.011383 77220 CGPG9271 SENSE NA NA 0.463 0.008 0.231 0.228 225 77334CGPG3638 SENSE NA NA 0.531 0.067 0.336 0.031 349 77349 CGPG8083 SENSE NANA 0.133 0.001 0.035 0.781 319 77517 CGPG6952 SENSE NA NA −0.843 0.142−1.458 0.062 320 77518 CGPG6953 SENSE NA NA 0.354 0.007 0.380 0.010 30777609 CGPG6326 SENSE NA NA 0.252 0.087 0.211 0.145

For “seeding weight” and “leaf angle”, if p<0.05 and delta or risk scoremean >0, the transgenic plants showed statistically significant traitimprovement as compared to the reference. If p<0.2 and delta or riskscore mean >0, the transgenic plants showed a trend of trait improvementas compared to the reference with p<0.2.

For “petiole length”, if p<0.05 and delta <0, the transgenic plantsshowed statistically significant trait improvement as compared to thereference. If p<0.2 and delta <0, the transgenic plants showed a trendof trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as setforth in SEQ ID NO: 246, 295, 303, 325, or 375 showed enhanced shadetolerance by the second criteria as illustrated in Example 1L and 1M.

I. Early Plant Growth and Development Screen

This example sets forth a plate based phenotypic analysis platform forthe rapid detection of phenotypes that are evident during the first twoweeks of growth. In this screen, we were looking for genes that conferadvantages in the processes of germination, seedling vigor, root growthand root morphology under non-stressed growth conditions to plants. Thetransgenic plants with advantages in seedling growth and developmentwere determined by the seedling weight and root length at day 14 afterseed planting.

T2 seeds were plated on glufosinate selection plates and grown understandard conditions (˜100 uE/m²/s, 16 h photoperiod, 22° C. at day, 20°C. at night). Seeds were stratified for 3 days at 4° C. Seedlings weregrown vertically (at a temperature of 22° C. at day 20° C. at night).Observations were taken on day 10 and day 14. Both seedling weight androot length at day 14 were analyzed as quantitative responses accordingto example 1M.

A list recombinant DNA constructs that improve early plant growth anddevelopment illustrated in Table 11.

TABLE 11 Root length Root length Seedling weight PEP at day 10 at day 14at day 14 SEQ Construct Nomination Delta P- Delta P- Delta P- ID ID IDOrientation mean value mean value mean value 227 19774 CGPG3920 SENSE0.488 0.097 0.307 0.100 0.550 0.163 229 19956 CGPG3972 SENSE 0.248 0.0620.154 0.214 0.284 0.001 232 19973 CGPG4026 SENSE 0.326 0.047 0.066 0.5770.444 0.012 302 70812 CGPG607 SENSE 0.704 0.037 0.546 0.031 0.740 0.033236 70915 CGPG4058 SENSE 0.292 0.005 0.278 0.008 0.331 0.114 230 70948CGPG3990 SENSE 0.255 0.269 0.093 0.229 0.375 0.026 234 70950 CGPG4052SENSE 0.106 0.577 0.124 0.374 0.239 0.041 235 70962 CGPG4057 SENSE 0.1710.075 0.144 0.226 0.283 0.015 241 70963 CGPG4121 SENSE 0.149 0.057 0.1120.074 0.062 0.513 242 70994 CGPG4122 SENSE 0.198 0.063 0.121 0.114 0.2400.124 244 70995 CGPG4154 SENSE 0.132 0.052 0.101 0.006 0.198 0.045 24971328 CGPG4454 SENSE 0.209 0.136 0.251 0.122 0.445 0.093 268 72001CGPG5221 SENSE 0.183 0.060 0.142 0.042 0.195 0.149 274 72753 CGPG5540SENSE 0.113 0.045 0.053 0.348 0.081 0.730 282 73105 CGPG5656 SENSE 0.0460.577 0.138 0.057 0.298 0.142 292 73136 CGPG5764 SENSE 0.212 0.077 0.1330.027 0.330 0.037 265 73256 CGPG5194 SENSE 0.123 0.503 0.371 0.135 0.6230.069 313 74420 CGPG6712 SENSE 0.249 0.085 0.093 0.358 0.363 0.042 31674566 CGPG6796 SENSE 0.169 0.003 0.147 0.114 0.173 0.171 304 74662CGPG6185 SENSE −0.205   0.337 0.009 0.878 −0.009 0.958 312 74688CGPG6653 SENSE 0.221 0.083 0.059 0.358 0.295 0.163 329 74862 CGPG7308SENSE 0.201 0.122 0.044 0.515 0.383 0.006 325 76178 CGPG7225 SENSE 0.2170.018 0.093 0.364 0.240 0.234 322 77069 CGPG7163 SENSE / / / / 0.6410.074 376 77124 CGPG9207 SENSE 0.337 0.121 0.256 0.045 0.762 0.034 37177155 CGPG9170 SENSE 0.181 0.262 0.265 0.035 0.419 0.000 367 77164CGPG9147 SENSE 0.589 0.068 0.369 0.066 0.899 0.051 370 77166 CGPG9163SENSE 0.383 0.025 0.391 0.006 0.787 0.019 372 77180 CGPG9180 SENSE 0.4420.058 0.267 0.031 0.402 0.339 362 77186 CGPG9133 SENSE 0.448 0.022 0.3830.079 0.654 0.057 365 77187 CGPG9141 SENSE 0.453 0.043 0.227 0.127 0.6600.013 375 77195 CGPG9205 SENSE 0.221 0.114 0.188 0.160 0.259 0.064 38577209 CGPG9278 SENSE 0.360 0.092 0.181 0.203 0.594 0.047 382 77266CGPG9259 SENSE / / / / 0.470 0.020 384 77268 CGPG9275 SENSE 0.353 0.1110.170 0.244 0.386 0.041 378 77273 CGPG9220 SENSE 0.255 0.070 0.023 0.5860.510 0.141 297 77312 CGPG5927 SENSE / / / / 1.006 0.065 393 77409CGPG9345 SENSE 0.410 0.027 0.396 0.011 0.461 0.040 389 77430 CGPG9322SENSE 0.209 0.183 0.211 0.247 0.399 0.092 390 77432 CGPG9335 SENSE 0.3420.113 0.285 0.122 0.425 0.055 391 77433 CGPG9341 SENSE 0.204 0.120 0.2220.041 0.382 0.085 392 77444 CGPG9344 SENSE 0.185 0.068 0.227 0.003 0.2760.002 387 77451 CGPG9309 SENSE 0.266 0.097 0.208 0.042 0.014 0.981 38877452 CGPG9311 SENSE 0.286 0.021 0.182 0.123 0.284 0.201 327 77536CGPG7272 SENSE 0.121 0.389 0.147 0.026 0.384 0.027 342 77549 CGPG7933SENSE 0.349 0.006 0.207 0.032 0.383 0.022 344 77568 CGPG8012 SENSE 0.3120.020 0.157 0.015 0.359 0.071 351 77587 CGPG8107 SENSE 0.151 0.128 0.2230.014 0.370 0.030 318 77618 CGPG6810 SENSE 0.179 0.018 0.104 0.276 0.1540.388 334 77816 CGPG7529 SENSE 0.145 0.400 0.171 0.142 0.312 0.181 33677821 CGPG7737 SENSE 0.114 0.406 0.126 0.208 0.311 0.079 343 77917CGPG7986 SENSE 0.136 0.083 0.162 0.011 −0.242 0.458

If p<0.05 and delta or risk score mean >0, the transgenic plants showedstatistically significant trait improvement as compared to thereference. If p<0.2 and delta or risk score mean >0, the transgenicplants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as setforth in SEQ ID NO: 252, 309, or 310 showed improved early plant growthand development evidenced by the second criteria as illustrated inExample 1L and 1M.

J. Late Plant Growth and Development Screen

This example sets forth a soil based phenotypic platform to identifygenes that confer advantages in the processes of leaf development,flowering production and seed maturity to plants.

Arabidopsis plants were grown on a commercial potting mixture (Metro Mix360, Scotts Co., Marysville, Ohio) consisting of 30-40% medium gradehorticultural vermiculite, 35-55% sphagnum peat moss, 10-20% processedbark ash, 1-15% pine bark and a starter nutrient charge. Soil wassupplemented with Osmocote time-release fertilizer at a rate of 30mg/ft³. T2 seeds were imbibed in 1% agarose solution for 3 days at 4° C.and then sown at a density of ˜5 per 2½″ pot. Thirty-two pots wereordered in a 4 by 8 grid in standard greenhouse flat. Plants were grownin environmentally controlled rooms under a 16 h day length with anaverage light intensity of ˜200 μmoles/m²/s. Day and night temperatureset points were 22° C. and 20° C., respectively. Humidity was maintainedat 65%. Plants were watered by sub-irrigation every two days on averageuntil mid-flowering, at which point the plants were watered daily untilflowering was complete.

Application of the herbicide glufosinate was performed to select T2individuals containing the target transgene. A single application ofglufosinate was applied when the first true leaves were visible. Eachpot was thinned to leave a single glufosinate-resistant seedling ˜3 daysafter the selection was applied.

The rosette radius was measured at day 25. The silique length wasmeasured at day 40. The plant parts were harvested at day 49 for dryweight measurements if flowering production was stopped. Otherwise, thedry weights of rosette and silique were carried out at day 53. The seedswere harvested at day 58. All measurements were analyzed as quantitativeresponses according to example 1M.

A list of recombinant DNA constructs that improve late plant growth anddevelopment illustrated in Table 12.

TABLE 12 Rosette Seed net Silique dry weight Rosette radius dry weightdry weight Silique length PEP at day 53 at day 25 at day 62 at day 53 atday 40 SEQ Construct Delta P- Delta P- Delta P- Delta P- Delta P- ID IDmean value mean value mean value mean value mean value 394 12313 −0.0750.489 0.654 0.000 0.157 0.174 −0.024 0.275 221 18258 −0.494 0.017 −0.1830.083 0.533 0.006 −0.269 0.234 0.056 0.009 246 70657 0.373 0.040 0.1870.037 0.355 0.081 −0.187 0.222 −0.003 0.972 247 70660 −0.010 0.936−0.072 0.431 0.445 0.026 0.116 0.369 0.145 0.045 216 71538 −0.167 0.1680.017 0.686 0.521 0.005 −0.196 0.113 0.023 0.557 269 72056 0.563 0.008NA NA −0.251 0.159 −0.289 0.314 −0.046 0.314 279 73057 −0.266 0.0350.167 0.114 0.396 0.034 −0.304 0.085 0.023 0.543 311 73568 −0.140 0.115−0.085 0.340 0.553 0.019 0.344 0.010 0.040 0.458 222 19193 −0.296 0.1100.081 0.385 1.076 0.011 NA NA 0.073 0.001 262 73242 0.159 0.132 0.1330.050 1.074 0.009 0.652 0.002 0.045 0.048 309 73433 0.477 0.026 0.1130.120 1.140 0.006 0.552 0.016 −0.009 0.872 316 74566 −0.387 0.023 0.1040.047 1.213 0.003 −0.169 0.130 0.012 0.598 338 75654 0.604 0.012 −0.0060.899 −1.193 0.004 −0.258 0.057 −0.004 0.962 361 77150 0.611 0.003−0.098 0.115 −0.363 0.011 −0.398 0.203 0.001 0.990 327 77536 0.043 0.6920.089 0.040 0.725 0.015 0.419 0.016 0.075 0.105 334 77816 −0.324 0.0210.005 0.954 1.207 0.000 −0.038 0.679 0.039 0.378

If p<0.05 and delta or risk score mean >0, the transgenic plants showedstatistically significant trait improvement as compared to thereference. If p<0.2 and delta or risk score mean >0, the transgenicplants showed a trend of trait improvement as compared to the reference.

K. Low Nitrogen Tolerance Screen

Under low nitrogen conditions, Arabidopsis seedlings become chloroticand have less biomass. This example sets forth the limited nitrogentolerance screen to identify Arabidopsis plants transformed with thegene of interest that are altered in their ability to accumulate biomassand/or retain chlorophyll under low nitrogen condition.

T2 seeds were plated on glufosinate selection plates containing 0.5×N-Free Hoagland's T 0.1 mM NH₄NO₃ T 0.1% sucrose T 1% phytagel media andgrown under standard light and temperature conditions. At 12 days ofgrowth, plants were scored for seedling status (i.e., viable ornon-viable) and root length. After 21 days of growth, plants were scoredfor BASTA resistance, visual color, seedling weight, number of greenleaves, number of rosette leaves, root length and formation of floweringbuds. A photograph of each plant was also taken at this time point.

The seedling weight and root length were analyzed as quantitativeresponses according to example 1M. The number green leaves, the numberof rosette leaves and the flowerbud formation were analyzed asqualitative responses according to example 1L. The leaf color raw datawere collected on each plant as the percentages of five color elements(Green, DarkGreen, LightGreen, RedPurple, YellowChlorotic) using acomputer imaging system. A statistical logistic regression model wasdeveloped to predict an overall value based on five colors for eachplant.

A list of recombinant DNA constructs that improve low nitrogenavailability tolerance in plants illustrated in Table 13.

TABLE 13 Leaf color PEP Root length Risk Rosette weight SEQ ConstructNomination Delta P- score P- Delta P- ID ID ID Orientation mean valuemean value mean value 200 10422 CGPG117 ANTI- −0.435 0.008 1.311 0.0210.050 0.396 SENSE 206 12602 CGPG170 SENSE −0.004 0.941 −0.052 0.8950.115 0.048 202 13235 CGPG1288 SENSE −0.047 0.267 −0.085 0.864 0.1280.068 203 13411 CGPG1301 SENSE 0.323 0.202 −3.233 0.126 0.174 0.044 20113485 CGPG1226 ANTI- 0.311 0.096 1.162 0.038 0.241 0.019 SENSE 205 13846CGPG1542 ANTI- −0.267 0.190 1.506 0.004 0.176 0.281 SENSE 212 16610CGPG2499 SENSE −0.037 0.677 1.325 0.004 −0.004 0.918 211 17805 CGPG2457ANTI- −0.348 0.016 3.523 0.067 −0.105 0.116 SENSE 218 18231 CGPG3274SENSE −0.207 0.117 0.925 0.062 −0.093 0.094 224 18354 CGPG3534 SENSE0.104 0.070 −0.407 0.124 0.189 0.048 252 70684 CGPG4588 SENSE 0.3790.001 −3.676 0.003 0.328 0.030 223 71301 CGPG3528 SENSE −0.394 0.0271.269 0.042 −0.128 0.026 250 71329 CGPG4456 SENSE −0.169 0.230 0.4750.074 −0.365 0.337 258 72813 CGPG4977 SENSE −0.361 0.193 0.521 0.0760.013 0.120 309 73433 CGPG6429 SENSE 0.129 0.201 −0.091 0.794 0.1860.002 300 74349 CGPG5967 SENSE −0.139 0.019 0.592 0.080 0.045 0.080 31374420 CGPG6712 SENSE 0.612 0.043 −3.086 0.063 0.173 0.002 298 75237CGPG5941 SENSE −0.097 0.237 0.948 0.031 0.074 0.136 333 75379 CGPG7520SENSE 0.000 0.998 0.529 0.048 −0.017 0.755 335 75434 CGPG7636 SENSE−0.336 0.039 1.208 0.076 −0.109 0.107 341 75622 CGPG7833 SENSE −0.5030.001 1.776 0.007 0.096 0.497 339 75692 CGPG7823 SENSE −0.055 0.165−0.377 0.328 0.132 0.083 301 76422 CGPG6040 SENSE 0.197 0.178 −0.2630.734 0.162 0.094 359 76961 CGPG9080 SENSE −0.361 0.136 1.472 0.013−0.190 0.066 369 77165 CGPG9155 SENSE 0.366 0.041 −1.982 0.143 0.2530.099 303 77322 CGPG6178 SENSE / / 1.691 0.060 −0.185 0.052 207 16322CGPG1828 SENSE / / −0.021 0.982 0.116 0.043 356 77832 CGPG8976 SENSE / /2.472 0.011 −0.028 0.445

For leaf color and rosette weight, if p<0.05 and delta or risk scoremean >0, the transgenic plants showed statistically significant traitimprovement as compared to the reference. If p<0.2 and delta or riskscore mean >0, the transgenic plants showed a trend of trait improvementas compared to the reference with p<0.2. For root length, if p<0.05, thetransgenic plants showed statistically significant trait improvement ascompared to the reference. If p<0.2, the transgenic plants showed atrend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as setforth in SEQ ID NO: 198 or 327 showed improved tolerance to low nitrogencondition evidenced by the second criteria as illustrated in Example 1Land 1M.

L. Statistic Analysis for Qualitative Responses

A list of responses that were analyzed as qualitative responsesillustrated in Table 14.

TABLE 14 categories response screen (success vs. failure) Wiltingresponse Soil drought tolerance non-wilted vs. wilted Risk Score screengrowth stage at heat stress tolerance 50% of plants reach day 14 screenstage1.03 vs. not growth stage at salt stress tolerance 50% of plantsreach day 14 screen stage1.03 vs. not growth stage at PEG inducedosmotic stress 50% of plants reach day 14 tolerance screen stage1.03 vs.not growth stage at cold germination tolerance 50% of plants reach day 7screen stage 0.5 vs. not number of rosette Shade tolerance screen 5leaves appeared leaves at day 23 vs. not Flower bud Shade tolerancescreen flower buds appear formation at day 23 vs. not leaf angle at day23 Shade tolerance screen >60 degree vs. <60 degree number of greenlimited nitrogen tolerance 6 or 7 leaves appeared leaves at day 21screen vs. not number of rosette limited nitrogen tolerance 6 or 7leaves appeared leaves at day 21 screen vs. not Flower bud limitednitrogen tolerance flower buds appear formation at day 21 screen vs. not

Plants were grouped into transgenic and reference groups and were scoredas success or failure according to Table 14. First, the risk (R) wascalculated, which is the proportion of plants that were scored as offailure plants within the group. Then the relative risk (RR) wascalculated as the ratio of R (transgenic) to R (reference). Risk score(RS) was calculated as −log₂ ^(RR). Two criteria were used to determinea transgenic with enhanced trait(s). Transgenic plants comprisingrecombinant DNA disclosed herein showed trait enhancement according toeither or both of the two criteria.

For the first criteria, the risk scores from multiple events of thetransgene of interest to were evaluated for statistical significance byt-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SASInstitute Inc, Cary, N.C., USA). RS with a value greater than 0indicates that the transgenic plants perform better than the reference.RS with a value less than 0 indicates that the transgenic plants performworse than the reference. The RS with a value equal to 0 indicates thatthe performance of the transgenic plants and the reference don't showany difference. If p<0.05 and risk score mean >0, the transgenic plantsshowed statistically significant trait enhancement as compared to thereference. If p<0.2 and risk score mean >0, the transgenic plants showeda trend of trait enhancement as compared to the reference.

For the second criteria, the RS from each event was evaluated forstatistical significance by t-test using SAS statistical software (SAS9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). The RSwith a value greater than 0 indicates that the transgenic plants fromthis events perform better than the reference. The RS with a value lessthan 0 indicates that the transgenic plants from this event performworse than the reference. The RS with a value equal to 0 indicates thatthe performance of the transgenic plants from this event and thereference don't show any difference. If p<0.05 and risk score mean >0,the transgenic plants from this event showed statistically significanttrait enhancement as compared to the reference. If p<0.2 and risk scoremean >0, the transgenic plants showed a trend of trait enhancement ascompared to the reference. If two or more events of the transgene ofinterest showed improvement in the same response, the transgene wasdeemed to show trait enhancement.

M. Statistic Analysis for Quantitative Responses

A list of responses that were analyzed as quantitative responsesillustrated in Table 15.

TABLE 15 response screen seed yield Soil drought stress tolerance screenseedling weight at day 14 heat stress tolerance screen root length atday 14 heat stress tolerance screen seedling weight at day 14 saltstress tolerance screen root length at day 14 salt stress tolerancescreen root length at day 11 salt stress tolerance screen seedlingweight at day 14 PEG induced osmotic stress tolerance screen root lengthat day 11 PEG induced osmotic stress tolerance screen root length at day14 PEG induced osmotic stress tolerance screen rosette area at day 8cold shock tolerance screen rosette area at day 28 cold shock tolerancescreen difference in rosette area cold shock tolerance screen from day 8to day 28 root length at day 28 cold germination tolerance screenseedling weight at day 23 Shade tolerance screen petiole length at day23 Shade tolerance screen root length at day 14 Early plant growth anddevelopment screen Seedling weight at day 14 Early plant growth anddevelopment screen Rosette dry weight at day 53 Late plant growth anddevelopment screen rosette radius at day 25 Late plant growth anddevelopment screen seed dry weight at day 58 Late plant growth anddevelopment screen silique dry weight at day 53 Late plant growth anddevelopment screen silique length at day 40 Late plant growth anddevelopment screen Seedling weight at day 21 Limited nitrogen tolerancescreen Root length at day 21 Limited nitrogen tolerance screen

The measurements (M) of each plant were transformed by log₂ calculation.The Delta was calculated as log₂ M(transgenic)−log₂ M(reference). Twocriteria were used to determine trait enhancement. A transgene ofinterest could show trait enhancement according to either or both of thetwo criteria. The measurements (M) of each plant were transformed bylog₂ calculation. The Delta was calculated as log₂ M(transgenic)−log₂M(reference). If the measured response was Petiole Length for the LowLight assay, Delta was subsequently multiplied by −1, to account for thefact that a shorter petiole length is considered an indication of traitenhancement.

For the first criteria, the Deltas from multiple events of the transgeneof interest were evaluated for statistical significance by t-test usingSAS statistical software (SAS 9, SAS/STAT User's Guide, SAS InstituteInc, Cary, N.C., USA). Delta with a value greater than 0 indicates thatthe transgenic plants perform better than the reference. Delta with avalue less than 0 indicates that the transgenic plants perform worsethan the reference. The Delta with a value equal to 0 indicates that theperformance of the transgenic plants and the reference don't show anydifference. If p<0.05 and risk score mean >0, the transgenic plantsshowed statistically significant trait enhancement as compared to thereference. If p<0.2 and risk score mean >0, the transgenic plants showeda trend of trait enhancement as compared to the reference.

For the second criteria, the delta from each event was evaluated forstatistical significance by t-test using SAS statistical software (SAS9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). The Deltawith a value greater than 0 indicates that the transgenic plants fromthis event performs better than the reference. The Delta with a valueless than 0 indicates that the transgenic plants from this event performworse than the reference. The Delta with a value equal to 0 indicatesthat the performance of the transgenic plants from this event and thereference don't show any difference. If p<0.05 and delta mean >0, thetransgenic plants from this event showed statistically significant traitimprovement as compared to the reference. If p<0.2 and delta mean >0,the transgenic plants showed a trend of trait enhancement as compared tothe reference. If two or more events of the transgene of interest showedenhancement in the same response, the transgene was deemed to show traitimprovement.

Example 2 Identification of Homologs

A BLAST searchable “All Protein Database” is constructed of knownprotein sequences using a proprietary sequence database and the NationalCenter for Biotechnology Information (NCBI) non-redundant amino aciddatabase (nr.aa). For each organism from which a DNA sequence providedherein was obtained, an “Organism Protein Database” is constructed ofknown protein sequences of the organism; the Organism Protein Databaseis a subset of the All Protein Database based on the NCBI taxonomy IDfor the organism.

The All Protein Database is queried using amino acid sequence of cognateprotein for gene DNA used in trait-improving recombinant DNA, i.e.,sequences of SEQ ID NO: 198 through SEQ ID NO: 394 using “blastp” withE-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated byorganism names. For each organism other than that of the query sequence,a list is kept for hits from the query organism itself with a moresignificant E-value than the best hit of the organism. The list containslikely duplicated genes, and is referred to as the Core List. Anotherlist was kept for all the hits from each organism, sorted by E-value,and referred to as the Hit List.

The Organism Protein Database is queried using amino acid sequences ofSEQ ID NO: 198 through SEQ ID NO: 394 using “blastp” with E-value cutoffof 1e-4. Up to 1000 top hits are kept. A BLAST searchable database isconstructed based on these hits, and is referred to as “SubDB”. SubDBwas queried with each sequence in the Hit List using “blastp” withE-value cutoff of 1e-8. The hit with the best E-value is compared withthe Core List from the corresponding organism. The hit is deemed alikely ortholog if it belongs to the Core List, otherwise it is deemednot a likely ortholog and there is no further search of sequences in theHit List for the same organism. Likely orthologs from a large number ofdistinct organisms were identified and are reported by amino acidsequences of SEQ ID NO: 395 to SEQ ID NO: 19938. These orthologs arereported in Tables 2 as homologs to the proteins cognate to genes usedin trait-improving recombinant DNA.

TABLE 2 SEQ ID NO: homolog SEQ ID NOs 198: 3331 2914 8408 6836 1084514799 13562 8848 2509 13003 875 10945 4176 7318 4820 19156 19137 1494217166 13809 3506 3410 1343 8019 7882 6490 3550 4999 3130 12635 877410864 17857 18089 16263 7295 10882 18252 3728 14833 11485 15463 188262924 18723 1446 2812 7847 3395 4209 15768 10679 18379 14040 3974 108711574 6329 5482 5609 6441 5537 17042 4716 4516 12643 11384 12736 81371344 19473 8452 4094 2460 18071 16891 5620 2364 7018 5200 11131 1813312170 2731 1687 5014 18511 9343 19283 6682 8123 8124 10597 10622 1060810600 16221 4280 11494 4867 9035 9507 7969 19159 19425 5248 17367 81358773 1287 2671 9365 18729 13828 8754 7170 14529 16604 8341 1135 62249739 11212 12962 4331 13555 14356 14357 14783 7509 2032 4692 5243 199:15320 200: 7710 14519 16336 7019 7872 7719 18299 10453 4650 15008 10089699 15645 10533 11751 12924 10526 5313 17532 8825 6941 7743 4155 145419797 16905 8304 12080 19803 3087 1397 7534 5547 2423 201: 11777 64441416 4353 15071 3413 14526 202: 19073 6651 15740 7399 15791 2977 77899248 14697 15923 12131 5470 965 11100 11069 12624 12892 3580 5270 186632916 1684 8107 4240 19240 18582 19054 15775 7301 17416 10566 16351 1384616386 18431 4439 4056 8159 8292 12484 7316 3763 4783 18427 15501 161647762 4015 4556 18955 12061 8636 9665 9713 10212 17555 5330 12850 1643619045 16034 16692 2571 11676 12666 11853 5348 8771 12218 3157 10749 203:5608 2382 17993 16598 7505 6976 204: 11369 13527 10897 6893 6962 11592837 5116 19669 1655 7244 2623 2591 16504 17797 4636 15133 4141 7112 79396898 15965 16706 16979 11784 16115 9620 15750 14652 3008 7838 14787 45631873 15811 7271 4317 16603 1715 14288 11341 6188 1546 5683 5225 30336069 11268 15981 15490 4181 8633 6058 7993 6763 9671 14892 17296 9908184 2935 8536 11232 12598 12638 5112 10787 1107 13858 5864 16398 1638017071 16520 1973 5455 13852 19228 1695 17402 2865 17120 2507 5601 149521462 6616 6973 3092 1382 7755 3870 9765 5711 1257 6119 15310 3792 1597016032 14584 7282 15140 7273 3013 15249 13253 13241 2265 17700 18976 400917354 2223 17559 3151 6320 17010 8939 11258 734 573 15015 3050 163443931 11238 7691 11499 1112 4825 8344 13519 11669 5640 13588 5473 151087210 17617 5994 5980 16627 17906 5876 6079 18894 6956 12887 14694 109681061 2778 6096 10570 680 974 3120 16300 5885 7626 13881 4244 4233 150394486 3103 3115 7654 11890 12359 10800 9409 12657 8977 9182 17818 29429780 9899 18496 4118 18188 10437 13907 17312 15730 13906 2603 12770 483712953 16663 9237 9546 16293 18238 6022 13908 10266 9180 5538 15536 1037015958 5217 8154 13095 15972 14841 15262 12702 10766 15960 18726 1939510345 5964 13064 3946 17225 17262 17220 17266 17224 17252 5465 549512800 12814 12758 3021 12400 12492 16461 13638 5616 17449 8397 172585669 5675 6852 9796 17601 17569 9775 17664 205: 14023 6117 12539 669618601 1463 19893 6621 9577 9166 17890 17889 528 5413 18313 2784 81023988 890 19106 571 6791 2402 912 206: 17912 8799 6566 12137 4356 58035253 6078 9123 17523 12392 17806 6917 19039 7350 17767 11311 17082 1553519392 11233 470 8318 14963 16736 8867 14135 5246 4674 2124 9901 1330215459 18997 2954 1026 667 7561 16841 8092 9084 4065 1836 13713 1369717640 7422 19580 4565 15193 526 13108 7065 11703 11362 3633 19430 622117433 15616 10082 10189 3089 15238 6633 8106 16180 15317 16364 207:14575 8831 11211 14985 17619 18829 2521 5219 15413 15835 1204 6569 46614457 6066 14578 932 13694 7541 4813 15707 19563 1326 16107 5009 751814825 13777 6175 15925 13617 5765 14838 9091 10776 15679 5909 19373 756208: 19581 18655 16310 7497 6382 8261 936 7358 8883 4866 2146 8601 29321626 10354 15192 11509 9909 13503 488 17477 14483 16941 2824 1072 498914179 4883 13751 209: 3493 16588 7031 5155 11150 17294 5241 10621 57865591 16509 2021 1324 12454 15061 1670 15143 16506 17002 8741 18091 40135959 19370 4239 12885 12174 3709 3431 9596 3378 1018 5963 13212 31502952 1619 19861 19114 15446 19582 12663 19603 210: 17777 843 11020 133718495 1493 6396 15336 14087 2568 4517 1157 10741 4553 14999 425 425817388 5327 748 11691 10174 17805 16628 10117 7980 8902 211: 9637 1726113232 7387 10506 19067 12021 872 11620 13603 841 1450 2848 16911 212:14398 1558 18339 13428 13431 4182 16260 10619 6127 12005 7704 5064 130724395 9032 19907 9588 478 12782 9917 13100 5202 10562 2044 4452 107753543 14840 14071 14020 10406 14218 1797 16955 3879 10312 12432 148247082 14554 15744 1508 15866 16232 6797 7077 5816 3597 3596 3311 15022881 2638 906 12037 12039 3458 2270 8812 3217 9466 18102 610 9042 243118349 16650 14418 2251 4235 6826 1244 16651 13004 7852 14435 9553 926215603 4340 9235 1413 10538 8296 17223 16048 16071 2120 19847 10420 1123918968 2995 7551 15854 12345 5685 18180 2381 4814 2159 16862 8677 1346611435 6649 6015 8097 11230 16025 9440 1353 16151 16249 15930 11226 93982870 12373 15043 11765 10145 11071 285 9270 9537 9095 2350 13362 54967924 11756 2624 7967 6715 15074 17068 9275 10177 11007 14857 8951 1218211643 13590 7684 1740 929 910 9093 10018 13344 16978 17544 6305 113781393 14108 15937 16631 11803 13126 11969 18524 9644 8425 8421 8420 1075510757 16696 15994 1143 3549 5868 5126 7506 12547 12669 19032 4707 47104708 4732 4736 4733 8772 4730 4711 3385 1831 2528 2304 15098 13223 1519711673 4386 11884 2336 14693 9118 9633 14548 12725 2399 18359 11011 146925652 5434 9755 9513 2681 4364 12903 19777 13525 4326 6859 6088 116113068 13940 3578 14440 8582 9826 19618 18012 17233 10057 18222 15021 93715985 14888 14001 1279 7826 14232 18172 7425 11502 15565 17431 8317 1029417742 5564 5531 18144 7400 10835 14389 13461 13857 7113 16444 4722 21157276 18254 1237 5256 13951 2920 16719 1045 15371 19022 684 8004 278119811 19766 3279 18712 4940 14312 6426 10076 15772 7906 19836 1121918985 2673 3933 14263 11860 1907 4672 15599 13737 4944 2252 15415 115433433 7746 17114 4115 4881 9445 14696 9525 14484 16112 5392 10428 14009349 7616 18930 2533 12585 10497 10236 18415 4874 19793 4633 12544 199517255 6130 10439 15261 6768 15985 17752 8504 12863 7487 14875 12020 203618127 9264 3628 5019 7498 15865 1448 9636 2840 3161 6207 2644 1059 206510031 2171 16927 4747 18701 19271 4174 1389 9893 12527 9018 10035 503910698 13943 7894 12671 5651 19586 12810 1254 3519 16114 4286 13948 144938294 14606 2586 14069 7712 16988 737 16797 16826 18966 5145 17610 63048895 7744 9280 9284 10647 16558 213: 12662 5295 19093 10971 15359 116318513 889 2151 3536 7105 3292 8466 18205 3579 1504 19486 13768 180476871 7174 11029 11505 17782 5697 18822 4892 12656 1284 3062 4697 599318343 18790 3057 7014 12265 10981 15204 11631 17704 17941 10633 19776933 10363 8446 17800 9822 2953 13693 19862 14551 15692 17673 6081 504912581 6910 214: 15345 19484 4871 3871 6695 3993 11459 9199 15899 1382114206 19489 13765 712 19072 11649 215: 1607 15669 13168 19044 790 95154511 13776 11573 13000 216: 4929 17143 10058 17506 15064 3332 5268 696816252 15949 6180 3000 14937 18454 13071 217: 6807 19716 480 14021 417912236 6337 12237 6338 6336 12234 4815 2261 18555 4706 7836 9176 188513443 9382 9439 8870 16691 16646 11516 11500 11515 11498 16670 1666016683 11513 11496 16668 16664 11519 16686 11479 11481 7146 16643 166594850 17951 7144 4824 7163 4851 11234 4860 17956 7160 17977 4827 48264859 7147 11475 7165 17928 17958 4853 11473 7143 11477 5129 18893 1891417526 17529 17548 17547 17549 18917 13686 19089 13683 4938 16999 136664494 12074 17097 12093 16874 12095 15878 17159 17154 17156 17153 568115126 10052 19712 4288 15399 742 11607 15654 7006 8041 715 2713 115227695 3581 7865 3776 15738 19821 17259 3948 13388 17427 15523 14216 871115423 19060 16814 14298 18720 7885 3967 9710 3568 3573 3575 8503 1548612932 7870 989 3595 17756 9202 520 11205 14771 3574 2443 8036 1953714641 6739 15375 2400 6753 3336 218: 13099 6217 4454 4699 11292 47021554 6467 15339 15722 11035 12964 16317 13914 17486 18809 14651 41899581 14957 2871 1561 17618 3835 14955 3868 9158 13663 10042 7575 198246200 10522 3600 4064 14590 12731 7201 19572 9060 4035 6255 3457 113207986 8818 219: 19325 10450 4487 12312 2564 10096 5641 1947 4367 696418031 1012 11041 15078 12255 5761 5061 9832 16834 18348 220: 8238 1920718657 1358 2734 9320 13526 12631 17660 16548 15528 18501 18049 1527419470 221: 2724 17089 9639 9208 17722 6817 11848 18904 13673 2347 115853054 7486 6056 1290 14932 14930 12636 14230 1572 15708 18912 18177 169910460 2090 5583 18802 606 5929 5041 19148 14224 9144 2643 17144 1414214674 16275 222: 14820 18626 11591 649 3005 3037 5453 5430 14248 23714626 11661 9784 10560 16219 11699 223: 17739 13288 4139 15466 19773 7531277 14333 4211 14510 662 7930 18703 3268 19400 17533 2302 9078 224:13859 17816 12692 10417 7051 5444 12975 1611 7050 17105 5023 3397 1314914751 10564 7331 7329 11868 9210 9211 18860 18879 14568 17920 1449712723 19352 10587 225: 11265 17061 11261 17058 11267 17064 8014 80338018 508 8035 8038 8013 8017 3554 13411 12545 12528 11997 12566 1192212520 11958 12525 12522 12553 11961 11979 5030 3464 3910 18901 1460114600 840 15664 15668 15121 15142 15119 15686 15690 15657 15688 1514615164 15716 15148 15656 11191 13623 12732 5050 5029 5011 5055 5035 50365032 5081 5058 5013 5078 5026 5059 5077 5008 5053 5076 11939 11976 125412898 17207 5179 5115 13674 13423 16078 9689 11174 5252 17279 17277 111771867 1865 1823 1844 1842 1793 1824 1820 10671 3139 1123 5805 12934 127557504 1091 17904 17909 17926 17902 17938 17907 17929 10598 17935 179335298 17980 10601 5383 14050 6585 11379 16583 17978 17814 4838 17812 26333257 3189 774 12008 9988 14268 18392 12567 14920 12980 11959 7682 768513235 4127 3740 3766 3770 3769 3795 14966 457 8521 19464 4847 13386 502413387 13401 5957 6571 7932 8369 10496 10492 10490 10495 10517 7943 49584956 4923 4986 4961 4951 5007 4937 4934 4984 4933 4924 4921 4936 49794932 4981 4988 18200 18196 7176 4739 5479 8351 7177 12886 9521 646417681 14504 907 16793 1869 14509 8882 19727 8798 1197 3419 14168 1284615739 1115 14165 19715 492 8873 1119 497 1194 3251 1116 8876 13982 1286014430 451 14969 6102 4358 2650 2649 11790 12925 5882 19696 9551 386719796 18933 18929 833 4908 14217 4852 4858 16064 14208 10736 4886 1421416062 14172 10735 14210 10720 1885 14187 14190 14184 14207 14703 1470714709 14706 14390 7658 7659 7661 2582 13794 5204 17305 16541 8914 1695917283 9043 3924 2696 8656 6648 2675 18504 17311 6052 9425 9406 9410 94116051 9452 6033 15964 6267 6031 8489 8463 8865 9427 6268 8490 8493 1560215621 14616 15649 15652 15606 11723 17247 9423 15595 15624 16148 22407428 11722 11363 2225 2196 5480 15579 15626 16142 7413 5483 11741 1614516162 11725 5481 18072 10973 12451 12340 15511 12185 4348 12754 872214544 6171 10732 9492 19795 12165 15082 6617 9613 739 19520 2497 1400511128 18795 14331 14329 14334 16439 12070 4053 3942 2387 2391 2394 755754 2950 752 2377 19819 17119 7118 1360 2273 11458 13758 11215 1127411229 13178 5110 16239 12884 18765 16637 19693 16831 17824 10810 1539515420 17470 10005 16397 16394 14711 14728 15503 4401 801 12244 1229212308 12219 9033 16763 14392 16804 16785 9566 16806 14409 14391 167629027 9568 9029 16788 9009 16780 14410 16781 16782 16802 3330 3357 33269001 3358 5789 14387 14412 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5445 16272 10838 19127 19142 9628 10532 7251 8084 9179 10060 32333234 10382 15456 12062 12060 8373 598 11569 18041 16210 3876 5308 53104105 19053 17469 5866 18520 18518 17299 16592 13763 1649 2826 1088 524417633 8002 6325 8510 8509 17342 10265 8523 11182 2722 2708 14181 1782810012 8552 6241 10590 5676 10274 8661 3295 6576 8917 12079 16538 1132211319 15823 16815 16795 13353 14432 2003 1985 7807 7667 1870 12245 1501115010 7081 7327 7326 3951 11419 11420 5668 5402 2091 2088 10840 161949002 9003 8127 8141 1440 6106 6104 17126 17005 4082 9591 9590 1915 672510684 7300 3317 3316 9330 19487 19488 14152 14153 2122 1914 1911 1066514295 14309 2813 2489 17345 11755 18173 14628 16281 4570 426 12610 1720117057 16060 16058 9609 987 9610 15673 921 1086 18306 11336 2498 1708717085 12034 12035 14678 14675 1138 544 16271 12318 12812 4308 1937814598 14599 7369 18594 7648 17481 19008 14345 5166 7493 13726 13725 83843400 16200 7902 8410 8409 13137 16958 16957 15723 12257 12253 1597812973 4460 4459 13017 1570 1078 2531 15107 15091 8965 13199 13198 1920319218 2645 523 5806 17244 17241 9229 13301 12697 16182 14543 17648 7956541 7604 12160 14921 4194 19094 17637 18276 18272 14246 14245 6265 113506263 11351 1447 9984 5779 16703 16701 8272 8270 15033 637 11702 338213215 6987 17734 17485 17484 8448 11578 16065 14275 12748 19807 22799116 2296 2779 13325 13222 5611 2538 12956 15346 10824 1158 4300 182556983 15168 4680 19799 12284 6950 16092 3445 2799 10239 3660 12422 279718788 11987 9273 10359 2829 13626 19734 592 14588 3599 6112 6093 6110572 5844 11779 4108 3093 11517 11539 455 10947 11127 13214 3927 1119912891 15461 15460 7465 5719 5718 3367 13458 13456 1149 14244 6969 121389126 1661 15396 5031 16825 1467 3689 6716 6528 6526 8496 18877 379:16324 10027 16399 19626 19600 476 10737 6503 2637 5207 7908 18645 137578204 17032 1213 10125 9070 5372 15085 10478 7338 18023 9641 11812 1464218106 9561 2651 1666 5437 14594 5439 12210 6002 12290 5485 6003 1435314349 14351 10819 8846 9429 2453 10112 16543 3140 1146 1124 1121 154382354 380: 15042 683 16630 18404 5663 2482 10670 11782 18811 18709 1170019144 6755 9799 13818 6126 2455 8789 381: 17923 18890 5500 4771 67455378 3236 1210 11603 19433 19342 15300 17953 11454 9261 12841 11591 54155453 5430 8278 14564 6761 17927 18569 14781 10539 6214 3620 3528 178998748 12221 5504 5502 9489 6425 8958 382: 19132 10614 8139 10836 1604011545 7879 2100 1703 14547 383: 1189 16611 3586 4641 19633 17705 171508849 3552 7294 2029 6153 18596 384: 741 1633 5923 8765 16673 14609 36986449 2951 2093 11563 614 14593 14762 6397 7731 14290 17038 4311 14640385: 11357 13113 2101 9359 2343 5986 9703 12857 8854 17190 4889 1380812572 3386 7845 17983 4794 386: 1016 16046 10106 6272 16847 10542 150625576 4893 5448 15407 7831 16578 18278 2702 7215 9961 6250 3252 1859916433 3226 17007 387: 7223 8111 7867 1322 9479 3291 18674 5132 125383515 18852 16518 14682 5419 9781 388: 13217 19387 1319 13219 1333 23844322 7600 13280 5924 13688 2181 16877 16122 5091 927 6100 3686 389:11269 4678 13073 1755 1754 8654 3662 17371 3891 3973 2187 15246 184624802 3390 619 643 13183 4014 1542 10000 19057 6229 1262 14305 1425617590 5708 4877 3775 8462 390: 17182 3673 6158 5731 8195 15335 7264 72675346 10728 11675 2337 19110 7961 8491 12046 10224 6729 18918 18228 31538723 3309 3313 6222 15956 4632 12325 6835 6387 2622 4496 9897 10860 804410003 13262 13236 6403 5331 4640 1303 4602 1304 18435 16053 18480 1764617644 17675 16363 4781 3269 8610 10852 3582 5717 9242 9340 9346 132711345 3780 11086 14006 2264 5062 5146 16786 891 1282 11639 2472 962119732 15665 10462 16384 18138 9510 19888 19119 4931 18991 13945 1893713580 18401 13863 1936 4639 10245 760 13679 14995 17389 5259 10385 976315381 10901 10886 10888 12415 15437 8840 7871 4684 12209 17107 1120316435 8624 13410 8530 10422 11650 15110 13639 12675 13689 19825 466213277 5625 9279 3369 5976 1377 2144 4086 18044 16889 6767 18418 118598188 6500 16529 16528 13234 14259 18077 11012 10071 11825 16181 64793800 9154 19377 924 923 7361 4574 14380 13895 17168 14901 17969 1373517599 10270 4099 18973 3523 14337 8378 4490 18286 5203 2114 13850 91099096 7060 4161 6561 1211 9640 13790 19180 14796 5771 15355 14027 628212681 16502 4842 4262 4930 15500 16490 14450 18361 15929 1171 7624 1383615083 19541 1490 8022 9426 19755 13700 10435 8346 13719 8681 19368 1090619116 1509 3865 12853 3125 5889 8316 18315 9077 436 1977 8354 5002 502516553 5171 17152 9352 17908 9960 9963 4907 2079 14164 3744 3102 183754128 4129 14794 391: 5830 10547 4780 6670 1560 15309 12977 11581 1524310500 7299 19550 4614 11584 18988 17503 13880 18201 7124 6036 1735915414 19074 13696 4180 810 12806 13939 1259 2434 12029 5666 8718 75118491 15717 1795 4475 1738 627 5732 18253 8660 11107 16309 12868 429914698 14679 14502 8248 11990 9758 16167 9385 10043 10430 16906 120995791 16810 13759 8562 2921 8889 10315 14077 2682 18457 14406 12241 101945832 13548 15304 4676 392: 18452 9432 5303 14233 9970 7637 14980 152657581 3016 10412 19809 393: 19384 19382 682 6463 1021 6039 6041 6057 78987896 13532 13533 8037 9356 16887 16866 7150 7148 11880 11903 7084 70833381 13816 13817 12463 4530 2311 2312 10624 10623 15054 2256 2257 1401614018 8801 6140 6142 8800 6138 8357 8375 8360 3666 3668 11044 11022 94739494 5001 5000 4831 4836 18327 19232 5812 5815 9999 7941 7942 749 30797426 7451 8001 7999 8005 19635 4051 15885 12205 2860 786 784 15576 1557512310 12307 2306 3461 7640 10242 394: 12840 13577 8132 1535 5905 32113797 12558 6843 17949 15017 9231 1310 17041 6367 18764 8461 15888 68666864 7983 8980 10079 15489 11102 9933 7325 1749 8441 7720 17727

Example 3 Consensus Sequence Build

ClustalW program is selected for multiple sequence alignments of anamino acid sequence of SEQ ID NO: 198 and its homologs, through SEQ IDNO: 394 and its homologs. Three major factors affecting the sequencealignments dramatically are (1) protein weight matrices; (2) gap openpenalty; (3) gap extension penalty. Protein weight matrices availablefor ClustalW program include Blosum, Pam and Gonnet series. Thoseparameters with gap open penalty and gap extension penalty wereextensively tested. On the basis of the test results, Blosum weightmatrix, gap open penalty of 10 and gap extension penalty of 1 werechosen for multiple sequence alignment. The consensus sequence of SEQ IDNO: 227 and its 25 homologs were derived according to the proceduredescribed above and is displayed in FIG. 1.

Example 4 Identification of Amino Acid Domain by Pfam Analysis

This example illustrates the identification of amino acid domain by Pfamanalysis.

The amino acid sequence of the expressed proteins that were shown to beassociated with an enhanced trait were analyzed for Pfam protein familyagainst the current Pfam collection of multiple sequence alignments andhidden Markov models using the HMMER software in the appended computerlisting. The Pfam protein families for the proteins of SEQ ID NO: 198through 394 are shown in Table 16. The Hidden Markov model databases forthe identified patent families are also in the appended computer listingallowing identification of other homologous proteins and their cognateencoding DNA to enable the full breadth of the invention for a person ofordinary skill in the art. Certain proteins are identified by a singlePfam domain and others by multiple Pfam domains. For instance, theprotein with amino acids of SEQ ID NO: 207 is characterized by two Pfamdomains, i.e. “TFIIS_M” and “TFIIS_C”. See also the protein with aminoacids of SEQ ID NO: 213 which is characterized by three copies of thePfam domain “WD40”. In Table 16 “score” is the gathering score for theHidden Markov Model of the domain which exceeds the gathering cutoffreported in Table 17.

TABLE 16 PEP Pfam domain SEQ ID NO GENE ID name begin stop score E-value198 CGPG106 CMAS 69 306 −149.8 5.00E−05 198 CGPG106 Methyltransf_11 131229 109.1 1.20E−29 198 CGPG106 Methyltransf_12 131 227 54.6 3.10E−13 200CGPG117 SNF5 14 252 66.9 5.90E−17 202 CGPG1288 Cupin_3 57 131 130.83.40E−36 204 CGPG1458 Phosphorylase 180 958 1454.7 0 205 CGPG1542 LMBR111 488 715.1 4.40E−212 206 CGPG170 Metallophos 54 249 140.4 4.50E−39 207CGPG1828 TFIIS_M 206 327 203.5 4.50E−58 207 CGPG1828 TFIIS_C 338 37683.3 7.00E−22 209 CGPG2217 ORMDL 14 156 356.8 3.20E−104 210 CGPG2292Tim17 52 186 16.1 4.20E−05 210 CGPG2292 SAM_1 192 253 13.8 0.019 212CGPG2499 Glycolytic 4 339 588.8 4.50E−174 213 CGPG2653 WD40 135 172 40.36.20E−09 213 CGPG2653 WD40 235 271 40.7 4.60E−09 213 CGPG2653 WD40 276313 38 3.00E−08 214 CGPG2813 HLH 204 251 34.1 4.60E−07 215 CGPG3002eIF-4B 5 451 −50.3 1.90E−10 216 CGPG3154 C2 14 102 101.8 1.80E−27 216CGPG3154 C2 198 272 57.8 3.20E−14 217 CGPG3235 B_lectin 66 176 156.85.30E−44 217 CGPG3235 S_locus_glycop 189 315 227 3.90E−65 217 CGPG3235PAN_2 332 402 103.9 4.20E−28 217 CGPG3235 Pkinase_Tyr 488 757 75.62.10E−20 217 CGPG3235 Pkinase 488 757 109 1.30E−29 218 CGPG3274Allene_ox_cyc 74 253 401.6 1.10E−117 219 CGPG3275 Auxin_inducible 50 155135.6 1.30E−37 220 CGPG3363 zf-AN1 21 58 10.5 0.0033 220 CGPG3363 zf-AN1103 148 12.4 0.0019 221 CGPG3367 TFIIA 9 375 556 3.40E−164 222 CGPG3375Tryp_alpha_amyl 33 111 55.2 1.90E−13 224 CGPG3534 Ank 42 74 13.6 0.31224 CGPG3534 Ank 75 107 36.8 6.90E−08 224 CGPG3534 Ank 108 140 1.6 17224 CGPG3534 Pkinase_Tyr 160 413 202.4 9.90E−58 224 CGPG3534 Pkinase 160413 198.5 1.40E−56 225 CGPG3638 Ribonuclease_T2 30 219 367.3 2.20E−107226 CGPG3918 Metallophos 60 255 159.1 1.10E−44 229 CGPG3972 14-3-3 8 243501.1 1.20E−147 230 CGPG3990 zf-C3HC4 259 299 47.3 4.60E−11 231 CGPG3994Pkinase 554 802 207 4.10E−59 231 CGPG3994 Pkinase_Tyr 554 802 2335.90E−67 232 CGPG4026 CMAS 59 320 −174.5 0.003 232 CGPG4026Methyltransf_11 122 220 96.3 8.10E−26 232 CGPG4026 Methyltransf_12 122218 37 5.90E−08 232 CGPG4026 Sterol_MT_C 229 358 301.1 1.80E−87 233CGPG4048 Pkinase 21 275 342.1 8.80E−100 233 CGPG4048 Pkinase_Tyr 21 27565.1 1.20E−19 233 CGPG4048 NAF 304 364 104.5 2.80E−28 234 CGPG4052DUF298 127 242 222.4 9.20E−64 236 CGPG4058 Asp 99 437 −128.4 4.60E−06237 CGPG4069 adh_short 31 218 −16.7 4.80E−05 239 CGPG4088 SWIB 324 39996.4 7.80E−26 241 CGPG4121 Cyclin_N 58 190 133.7 4.80E−37 241 CGPG4121Cyclin_C 192 314 44.3 3.70E−10 242 CGPG4122 zf-C3HC4 100 141 27.64.10E−05 243 CGPG4140 p450 48 484 140.8 3.30E−39 244 CGPG4154 zf-CCCH 90115 22.6 0.0012 245 CGPG4311 FBPase 70 395 347.9 1.50E−101 247 CGPG4369BoIA 10 79 92.1 1.60E−24 248 CGPG442 2-Hacid_dh 85 394 140.9 3.20E−39248 CGPG442 2-Hacid_dh_C 187 362 293 5.20E−85 248 CGPG442 ACT 551 62139.9 8.10E−09 249 CGPG4454 p450 45 447 −35.5 2.10E−09 250 CGPG4456 p45075 502 103.6 5.30E−28 252 CGPG4588 Auxin_inducible 1 102 151.5 2.00E−42253 CGPG4765 DUF868 28 304 175.5 1.20E−49 255 CGPG4912 WD40 243 281 313.80E−06 256 CGPG4926 WD40 42 79 22.8 0.0012 256 CGPG4926 WD40 126 16325.8 0.00014 257 CGPG4967 Asp 71 424 −94.7 4.70E−08 258 CGPG4977 Usp 3157 85.3 1.80E−22 259 CGPG5001 adh_short 5 181 6.4 1.40E−06 260 CGPG5025adh_short 30 212 5.7 1.50E−06 261 CGPG5041 DUF26 77 132 84.3 3.40E−22261 CGPG5041 DUF26 188 242 100.7 4.00E−27 261 CGPG5041 Pkinase 333 55860.3 5.70E−15 262 CGPG5116 ArfGap 15 137 174.1 3.20E−49 262 CGPG5116 C2182 261 101.4 2.40E−27 263 CGPG5144 p450 61 535 157.9 2.40E−44 264CGPG5171 p450 35 496 137 4.60E−38 265 CGPG5194 DUF1191 25 308 616.22.60E−182 268 CGPG5221 p450 30 499 135.4 1.40E−37 269 CGPG5269 PCI 297401 108.9 1.30E−29 270 CGPG5404 Peptidase_S10 68 480 683.3 1.70E−202 271CGPG5432 MtN3_slv 12 99 145.4 1.40E−40 271 CGPG5432 MtN3_slv 133 219140.4 4.40E−39 272 CGPG5518 Ribosomal_S8e 1 238 325.9 6.30E−95 273CGPG5535 WD40 335 371 28.8 1.80E−05 273 CGPG5535 WD40 434 471 37.15.50E−08 273 CGPG5535 WD40 476 513 47.2 5.20E−11 273 CGPG5535 WD40 518555 35.6 1.60E−07 273 CGPG5535 WD40 567 604 36.1 1.10E−07 273 CGPG5535WD40 621 658 34.9 2.50E−07 273 CGPG5535 WD40 663 706 35.5 1.60E−07 274CGPG5540 ESCRT-III 21 207 265.7 8.40E−77 275 CGPG5568 AA_permease 69 521−67.1 0.00013 276 CGPG5577 SMC_N 21 1049 −2.8 1.50E−11 277 CGPG5587Thioredoxin 78 199 0.1 0.00015 278 CGPG5594 Histone 27 100 99.6 8.80E−27279 CGPG5633 PGAM 91 277 153.2 6.30E−43 280 CGPG5640 Aminotran_1_2 30385 362.1 8.00E−106 281 CGPG5646 iPGM_N 2 363 876.8 9.10E−261 281CGPG5646 Metalloenzyme 373 488 173.3 5.40E−49 282 CGPG5656 Gln-synt_N 1799 85.7 1.30E−22 282 CGPG5656 Gln-synt_C 105 358 528.1 8.50E−156 283CGPG5659 Aminotran_3 35 361 321.7 1.20E−93 284 CGPG5661 PK 1 345 7023.80E−208 284 CGPG5661 PK_C 355 469 115.4 1.50E−31 285 CGPG5684Glycolytic 4 339 578.6 5.60E−171 286 CGPG5694 TIM 4 246 465.9 4.50E−137287 CGPG5704 NDK 2 136 329 7.30E−96 288 CGPG5714 NDK 4 138 266.45.20E−77 289 CGPG5721 Rib_5-P_isom_A 47 215 354.4 1.70E−103 290 CGPG5728zf-CCCH 80 106 39.3 1.20E−08 291 CGPG5757 Sad1_UNC 203 330 195.61.00E−55 292 CGPG5764 Actin 11 140 36.4 6.60E−08 293 CGPG5783 TPT 272442 71.5 2.40E−18 294 CGPG5791 AA_permease 114 588 704.9 5.40E−209 295CGPG5799 Aa_trans 206 601 453 3.60E−133 296 CGPG5856 Pkinase 79 353159.5 8.00E−45 296 CGPG5856 Pkinase_Tyr 79 353 149.4 9.00E−42 297CGPG5927 AAA 58 248 252.4 8.70E−73 297 CGPG5927 AAA 322 510 290.82.40E−84 298 CGPG5941 PfkB 42 336 86.2 9.00E−23 299 CGPG5957 CBM_20 86178 23 5.70E−07 300 CGPG5967 DUF822 2 147 307.1 2.90E−89 301 CGPG6040LEA_3 1 88 178.7 1.30E−50 302 CGPG607 PurA 28 275 44.4 5.80E−12 303CGPG6178 DUF1336 53 267 427.9 1.30E−125 304 CGPG6185 UQ_con 7 148 197.92.10E−56 305 CGPG6306 APC8 1 161 401.5 1.10E−117 305 CGPG6306 TPR_1 339372 34.7 2.90E−07 305 CGPG6306 TPR_2 339 372 23.8 0.00058 305 CGPG6306TPR_2 373 406 23.7 0.00062 305 CGPG6306 TPR_1 373 406 34.1 4.40E−07 305CGPG6306 TPR_2 407 440 22 0.0019 305 CGPG6306 TPR_1 407 440 24.3 0.00039306 CGPG6318 MFS_1 31 496 35.3 1.90E−07 306 CGPG6318 PTR2 92 484 208.61.30E−59 307 CGPG6326 Kelch_1 34 79 45 2.40E−10 307 CGPG6326 Kelch_2 3479 43.5 6.80E−10 307 CGPG6326 Kelch_1 152 198 26.7 7.70E−05 307 CGPG6326Kelch_2 152 198 32.1 1.80E−06 307 CGPG6326 Kelch_2 203 249 20.2 0.0067307 CGPG6326 Kelch_1 204 248 7.9 0.45 308 CGPG6370 Gp_dh_N 3 151 3265.80E−95 308 CGPG6370 Gp_dh_C 156 313 362 8.80E−106 309 CGPG6429 ADH_N25 135 159.4 8.40E−45 309 CGPG6429 ADH_zinc_N 166 305 105.1 1.90E−28 310CGPG6440 PK 1 345 789.4 1.90E−234 310 CGPG6440 PK_C 357 471 167.53.10E−47 310 CGPG6440 PEP-utilizers 486 575 134 3.60E−37 311 CGPG6516Aldedh 19 478 778.3 4.10E−231 312 CGPG6653 LRRNT_2 23 66 49.3 1.20E−11312 CGPG6653 LRR_1 71 93 12.1 1.4 312 CGPG6653 LRR_1 95 117 10.5 2.8 312CGPG6653 LRR_1 119 142 13 0.93 312 CGPG6653 LRR_1 144 166 19.5 0.011 312CGPG6653 LRR_1 168 190 10.6 2.7 312 CGPG6653 LRR_1 192 214 8.8 5.7 312CGPG6653 LRR_1 289 311 17.4 0.046 312 CGPG6653 LRR_1 313 335 10.8 2.4312 CGPG6653 LRR_1 337 359 10.7 2.5 312 CGPG6653 LRR_1 361 384 12 1.5312 CGPG6653 LRR_1 409 431 10.4 2.9 312 CGPG6653 LRR_1 457 479 11.9 1.5312 CGPG6653 LRR_1 481 503 10.4 3 312 CGPG6653 LRR_1 505 527 10.5 2.8312 CGPG6653 LRR_1 529 551 11.1 2.2 312 CGPG6653 LRR_1 553 575 9.3 4.7312 CGPG6653 LRR_1 577 598 11.1 2.1 312 CGPG6653 Pkinase 695 966 134.82.20E−37 312 CGPG6653 Pkinase_Tyr 695 966 134.5 2.60E−37 313 CGPG6712PGAM 91 277 154.6 2.30E−43 314 CGPG6737 PGAM 92 253 173 6.80E−49 315CGPG6747 FBPase 106 429 448.6 7.20E−132 316 CGPG6796 Alpha-amylase 14452 199.5 7.00E−57 318 CGPG6810 GH3 12 561 1261.5 0 320 CGPG6953 Ank 121153 50.4 5.60E−12 321 CGPG7121 L51_S25_CI-B8 20 93 108.9 1.40E−29 322CGPG7163 Prenylcys_lyase 149 500 788 5.20E−234 324 CGPG7206 Aldo_ket_red14 298 389.4 4.80E−114 325 CGPG7225 Subtilisin_N 48 125 84.3 3.30E−22326 CGPG7267 DUF588 34 164 154.6 2.40E−43 327 CGPG7272 DUF1005 50 254524.8 8.50E−155 328 CGPG7281 FA_hydroxylase 86 229 361.9 9.30E−106 329CGPG7308 CoA_binding 1 100 −10.8 0.04 329 CGPG7308 NAD_Gly3P_dh_N 4 147−0.6 2.00E−06 329 CGPG7308 F420_oxidored 5 251 282.5 7.50E−82 330CGPG7316 Anti-silence 1 155 419.9 3.20E−123 331 CGPG7371 Response_reg 29157 94.8 2.30E−25 332 CGPG7457 PfkB 114 408 146.6 6.00E−41 335 CGPG7636LSM 13 81 78.2 2.40E−20 338 CGPG7804 FAR1 62 279 364.9 1.20E−106 338CGPG7804 SWIM 556 589 37.3 4.90E−08 339 CGPG7823 Rotamase 104 188 91.81.90E−24 339 CGPG7823 Rhodanese 203 298 44.1 4.40E−10 340 CGPG7828 DnaJ12 81 66.1 1.00E−16 340 CGPG7828 zf-CSL 96 174 25.2 0.00021 343 CGPG7986F-box 48 96 31.2 3.40E−06 343 CGPG7986 LRR_1 189 216 9 5.4 343 CGPG7986LRR_1 428 451 8.2 7.7 343 CGPG7986 LRR_1 561 584 8.4 6.9 345 CGPG8015zf-CCHC 18 35 24.2 7.90E−05 349 CGPG8083 Tryp_alpha_amyl 28 105 37.63.90E−08 350 CGPG8106 BURP 56 280 380 3.20E−111 353 CGPG8152PAP_fibrillin 9 124 45.9 1.20E−10 354 CGPG8166 PBD 118 166 49.9 7.80E−12355 CGPG8377 Oleosin 30 109 55 2.20E−13 356 CGPG8976 Ceramidase_alk 50795 1545.5 0 357 CGPG8987 FH2 439 839 552.9 3.10E−163 358 CGPG9013NAD_binding_1 234 350 138.6 1.50E−38 359 CGPG9080 EGF_CA 315 357 42.21.60E−09 359 CGPG9080 Pkinase 433 716 120.3 4.90E−33 359 CGPG9080Pkinase_Tyr 433 704 113.1 7.40E−31 360 CGPG9081 DUF676 30 247 3197.50E−93 361 CGPG9130 MMR_HSR1 266 369 71.9 1.80E−18 362 CGPG9133 PPR125 159 13.2 0.19 362 CGPG9133 PPR 161 195 2.8 3.2 362 CGPG9133 PPR 196230 22.7 0.0012 362 CGPG9133 PPR 232 266 42.1 1.70E−09 362 CGPG9133 PPR267 301 29.4 1.20E−05 362 CGPG9133 PPR 302 336 49.4 1.10E−11 362CGPG9133 PPR 337 371 32.9 1.00E−06 362 CGPG9133 PPR 372 407 7.9 0.8 362CGPG9133 PPR 408 442 49.7 8.80E−12 362 CGPG9133 PPR 443 477 22.6 0.0013362 CGPG9133 PPR 478 512 37.8 3.40E−08 362 CGPG9133 PPR 513 547 42.91.00E−09 362 CGPG9133 PPR 548 582 30.2 6.70E−06 362 CGPG9133 PPR 583 61738.4 2.20E−08 363 CGPG9134 HD 91 232 46.2 1.00E−10 364 CGPG9137 RrnaAD65 337 122.2 1.30E−33 365 CGPG9141 Pantoate_transf 40 306 402.94.40E−118 366 CGPG9145 Lung_7-TM_R 168 423 385.2 8.80E−113 367 CGPG9147DNA_pol_E_B 178 389 249.9 5.00E−72 368 CGPG9148 p450 36 502 128.81.40E−35 369 CGPG9155 Pkinase 86 347 56.9 6.10E−14 369 CGPG9155Pkinase_Tyr 86 351 73.7 2.90E−20 370 CGPG9163 Na_H_Exchanger 12 378280.1 3.80E−81 370 CGPG9163 TrkA_N 416 531 118.8 1.50E−32 371 CGPG9170Complex1_30kDa 90 158 103.8 4.70E−28 371 CGPG9170 Complex1_49kDa 298 5372.8 6.10E−13 373 CGPG9183 HTH_11 1 56 72.4 1.30E−18 373 CGPG9183BPL_LipA_LipB 84 182 94.5 2.90E−25 373 CGPG9183 BPL_C 275 322 43.37.80E−10 374 CGPG9186 DHBP_synthase 8 203 370.6 2.20E−108 374 CGPG9186GTP_cyclohydro2 208 366 −2.3 3.80E−10 375 CGPG9205 NTP_transferase 4 288421 1.50E−123 375 CGPG9205 MannoseP_isomer 299 465 350.8 2.00E−102 375CGPG9205 Cupin_2 380 450 55.3 1.80E−13 376 CGPG9207 HTH_11 6 59 50.93.90E−12 376 CGPG9207 BPL_LipA_LipB 83 180 104.5 2.80E−28 376 CGPG9207BPL_C 271 317 46.5 8.50E−11 377 CGPG9219 Complex1_30kDa 107 175 99.87.60E−27 377 CGPG9219 Complex1_49kDa 311 537 −11.6 5.90E−12 378 CGPG9220GDC-P 3 443 700.9 8.30E−208 379 CGPG9230 Peptidase_S10 88 488 657.21.20E−194 381 CGPG9238 Tryp_alpha_amyl 36 114 56 1.20E−13 382 CGPG9259Mit_rib_S27 14 93 135.3 1.50E−37 383 CGPG9271 NPH3 215 418 189.95.60E−54 384 CGPG9275 ETC_C1_NDUFA5 35 91 112.8 9.00E−31 386 CGPG9283DUF1195 6 161 180.3 4.30E−51 387 CGPG9309 MAP65_ASE1 38 575 52.81.10E−12 389 CGPG9322 Pkinase 103 383 170 5.40E−48 389 CGPG9322Pkinase_Tyr 103 383 158.7 1.30E−44 390 CGPG9335 Sugar_tr 7 726 210.63.30E−60 390 CGPG9335 MFS_1 11 685 107.7 3.00E−29 391 CGPG9341 RMMBL 531573 35.9 1.20E−07 392 CGPG9344 TPR_2 531 564 23.9 0.00051 393 CGPG9345UPF0261 5 432 483.7 2.00E−142 394 CGPG976 Glyco_transf_8 216 533 401.31.30E−117

TABLE 17 Pfam domain accession gathering name number cutoff domaindescription 14-3-3 PF00244.9 25 14-3-3 protein 2-Hacid_dh PF00389.1911.2 D-isomer specific 2-hydroxyacid dehydrogenase, catalytic domain2-Hacid_dh_C PF02826.7 −82.2 D-isomer specific 2-hydroxyaciddehydrogenase, NAD binding domain AAA PF00004.18 12.3 ATPase familyassociated with various cellular activities (AAA) AA_permease PF00324.10−120.8 Amino acid permease ACT PF01842.13 0 ACT domain ADH_N PF08240.2−14.5 Alcohol dehydrogenase GroES-like domain ADH_zinc_N PF00107.16 23.8Zinc-binding dehydrogenase APC8 PF04049.3 −19.8 Anaphase promotingcomplex subunit 8/Cdc23 Aa_trans PF01490.7 −128.4 Transmembrane aminoacid transporter protein Actin PF00022.8 −30 Actin Aldedh PF00171.11−209.3 Aldehyde dehydrogenase family Aldo_ket_red PF00248.10 −97Aldo/keto reductase family Allene_ox_cyc PF06351.2 25 Allene oxidecyclase Alpha-amylase PF00128.12 −93 Alpha amylase, catalytic domainAminotran_1_2 PF00155.10 −57.5 Aminotransferase class I and IIAminotran_3 PF00202.10 −207.6 Aminotransferase class-III Ank PF00023.180 Ankyrin repeat Anti-silence PF04729.4 25 Anti-silencing protein,ASF1-like ArfGap PF01412.8 −17 Putative GTPase activating protein forArf Asp PF00026.13 −186.1 Eukaryotic aspartyl protease Auxin_induciblePF02519.4 −15 Auxin responsive protein BPL_C PF02237.6 16 Biotin proteinligase C terminal domain BPL_LipA_LipB PF03099.8 −0.2 Biotin/lipoate A/Bprotein ligase family BURP PF03181.5 −52 BURP domain B_lectin PF01453.1428.2 D-mannose binding lectin BolA PF01722.7 23 BolA-like protein C2PF00168.18 3.7 C2 domain CBM_20 PF00686.9 −3 Starch binding domain CMASPF02353.10 −177.9 Cyclopropane-fatty-acyl-phospholipid synthaseCeramidase_alk PF04734.3 25 Neutral/alkaline non-lysosomal ceramidaseCoA_binding PF02629.8 −12.8 CoA binding domain Complex1_30 kDa PF00329.8−3 Respiratory-chain NADH dehydrogenase, 30 Kd subunit Complex1_49 kDaPF00346.8 −108 Respiratory-chain NADH dehydrogenase, 49 Kd subunitCupin_2 PF07883.1 16.6 Cupin domain Cupin_3 PF05899.2 4.4 Protein ofunknown function (DUF861) Cyclin_C PF02984.8 −13 Cyclin, C-terminaldomain Cyclin_N PF00134.13 −14.7 Cyclin, N-terminal domain DHBP_synthasePF00926.10 −116 3,4-dihydroxy-2-butanone 4-phosphate synthaseDNA_pol_E_B PF04042.5 −47.5 DNA polymerase alpha/epsilon subunit BDUF1005 PF06219.2 25 Protein of unknown function (DUF1005) DUF1191PF06697.2 25 Protein of unknown function (DUF1191) DUF1195 PF06708.1 25Protein of unknown function (DUF1195) DUF1336 PF07059.2 −78.2 Protein ofunknown function (DUF1336) DUF26 PF01657.7 0 Domain of unknown functionDUF26 DUF298 PF03556.6 25 Domain of unknown function (DUF298) DUF588PF04535.2 25 Domain of unknown function (DUF588) DUF676 PF05057.4 −60.7Putative serine esterase (DUF676) DUF822 PF05687.3 25 Plant protein ofunknown function (DUF822) DUF868 PF05910.2 25 Plant protein of unknownfunction (DUF868) DnaJ PF00226.19 −8 DnaJ domain EGF_CA PF07645.4 24.5Calcium binding EGF domain ESCRT-III PF03357.10 −35.4 ESCRT-III complexsubunit ETC_C1_NDUFA5 PF04716.3 25 ETC complex I subunit conservedregion F-box PF00646.21 13.6 F-box domain F420_oxidored PF03807.6 −34.5NADP oxidoreductase coenzyme F420- dependent FAR1 PF03101.4 0 FAR1family FA_hydroxylase PF04116.2 −64.1 Fatty acid hydroxylase FBPasePF00316.10 −170.3 Fructose-1-6-bisphosphatase FH2 PF02181.13 −98.3Formin Homology 2 Domain GDC-P PF02347.5 −306.2 Glycine cleavage systemP-protein GH3 PF03321.3 −336 GH3 auxin-responsive promoterGTP_cyclohydro2 PF00925.11 −49 GTP cyclohydrolase II Gln-synt_CPF00120.14 −124 Glutamine synthetase, catalytic domain Gln-synt_NPF03951.9 9 Glutamine synthetase, beta-Grasp domain Glyco_transf_8PF01501.9 −43.2 Glycosyl transferase family 8 Glycolytic PF00274.9−174.5 Fructose-bisphosphate aldolase class-I Gp_dh_C PF02800.9 −64.1Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain Gp_dh_NPF00044.12 −74.2 Glyceraldehyde 3-phosphate dehydrogenase, NAD bindingdomain HD PF01966.11 18 HD domain HLH PF00010.15 8.2 Helix-loop-helixDNA-binding domain HTH_11 PF08279.1 11.3 HTH domain Histone PF00125.1317.4 Core histone H2A/H2B/H3/H4 Kelch_1 PF01344.14 7.8 Kelch motifKelch_2 PF07646.4 14 Kelch motif L51_S25_CI-B8 PF05047.5 25Mitochondrial ribosomal protein L51/S25/CI-B8 domain LEA_3 PF03242.3 25Late embryogenesis abundant protein LMBR1 PF04791.5 −116.3 LMBR1-likemembrane protein LRRNT_2 PF08263.2 18.6 Leucine rich repeat N-terminaldomain LRR_1 PF00560.21 7.7 Leucine Rich Repeat LSM PF01423.12 13.7 LSMdomain Lung_7-TM_R PF06814.3 25 Lung seven transmembrane receptorMAP65_ASE1 PF03999.2 −134.8 Microtubule associated protein (MAP65/ASE1family) MFS_1 PF07690.5 23.5 Major Facilitator Superfamily MMR_HSR1PF01926.11 31.2 GTPase of unknown function MannoseP_isomer PF01050.8 −70Mannose-6-phosphate isomerase Metalloenzyme PF01676.7 −14.4Metalloenzyme superfamily Metallophos PF00149.17 22 Calcineurin-likephosphoesterase Methyltransf_11 PF08241.1 17.1 Methyltransferase domainMethyltransf_12 PF08242.1 21.4 Methyltransferase domain Mit_rib_S27PF08293.1 25 Mitochondrial ribosomal subunit S27 MtN3_slv PF03083.5 −0.8MtN3/saliva family NAD_Gly3P_dh_N PF01210.12 −44 NAD-dependentglycerol-3-phosphate dehydrogenase N-terminus NAD_binding_1 PF00175.10−3.9 Oxidoreductase NAD-binding domain NAF PF03822.4 4.5 NAF domain NDKPF00334.9 −59.9 Nucleoside diphosphate kinase NPH3 PF03000.4 25 NPH3family NTP_transferase PF00483.12 −90.5 Nucleotidyl transferaseNa_H_Exchanger PF00999.10 −67.9 Sodium/hydrogen exchanger family ORMDLPF04061.4 25 ORMDL family Oleosin PF01277.7 −27 Oleosin PAN_2 PF08276.2−4.9 PAN-like domain PAP_fibrillin PF04755.2 25 PAP_fibrillin PBDPF00786.17 12.2 P21-Rho-binding domain PCI PF01399.15 25 PCI domainPEP-utilizers PF00391.12 10 PEP-utilising enzyme, mobile domain PGAMPF00300.12 −3 Phosphoglycerate mutase family PK PF00224.10 −244 Pyruvatekinase, barrel domain PK_C PF02887.5 −44 Pyruvate kinase, alpha/betadomain PPR PF01535.11 0 PPR repeat PTR2 PF00854.12 −50 POT familyPantoate_transf PF02548.5 −93 Ketopantoate hydroxymethyltransferasePeptidase_S10 PF00450.11 −198 Serine carboxypeptidase PfkB PF00294.13−67.8 pfkB family carbohydrate kinase Phosphorylase PF00343.9 −601.1Carbohydrate phosphorylase Pkinase PF00069.14 −70.8 Protein kinasedomain Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase Prenylcys_lyasePF07156.3 −164.1 Prenylcysteine lyase PurA PF04845.3 25 PurA ssDNA andRNA-binding protein RMMBL PF07521.1 18.5 RNA-metabolisingmetallo-beta-lactamase Response_reg PF00072.12 4 Response regulatorreceiver domain Rhodanese PF00581.9 25 Rhodanese-like domainRib_5-P_isom_A PF06026.4 25 Ribose 5-phosphate isomerase A(phosphoriboisomerase A) Ribonuclease_T2 PF00445.8 −53 Ribonuclease T2family Ribosomal_S8e PF01201.11 25 Ribosomal protein S8e RotamasePF00639.10 4 PPIC-type PPIASE domain RrnaAD PF00398.9 −73.3 RibosomalRNA adenine dimethylase SAM_1 PF00536.18 11.3 SAM domain (Sterile alphamotif) SMC_N PF02463.8 −95.8 RecF/RecN/SMC N terminal domain SNF5PF04855.3 25 SNF5/SMARCB1/INI1 SWIB PF02201.9 −7 SWIB/MDM2 domain SWIMPF04434.7 10 SWIM zinc finger S_locus_glycop PF00954.11 −12.7 S-locusglycoprotein family Sad1_UNC PF07738.2 −20.4 Sad1/UNC-like C-terminalSterol_MT_C PF08498.1 30.5 Sterol methyltransferase C-terminalSubtilisin_N PF05922.6 26.1 Subtilisin N-terminal Region Sugar_trPF00083.13 −85 Sugar (and other) transporter TFIIA PF03153.4 25Transcription factor IIA, alpha/beta subunit TFIIS_C PF01096.9 15Transcription factor S-II (TFIIS) TFIIS_M PF07500.3 7.4 Transcriptionfactor S-II (TFIIS), central domain TIM PF00121.8 −97 Triosephosphateisomerase TPR_1 PF00515.16 7.7 Tetratricopeptide repeat TPR_2 PF07719.520.1 Tetratricopeptide repeat TPT PF03151.7 −15.3 Triose-phosphateTransporter family Thioredoxin PF00085.9 −25.7 Thioredoxin Tim17PF02466.8 2.7 Tim17/Tim22/Tim23 family TrkA_N PF02254.7 4.7 TrkA-Ndomain Tryp_alpha_amyl PF00234.10 −4 Protease inhibitor/seed storage/LTPfamily UPF0261 PF06792.1 25 Uncharacterised protein family (UPF0261)UQ_con PF00179.16 −30 Ubiquitin-conjugating enzyme Usp PF00582.16 36.1Universal stress protein family WD40 PF00400.20 21.5 WD domain, G-betarepeat adh_short PF00106.14 −17 short chain dehydrogenase eIF-4BPF06273.1 −205.7 Plant specific eukaryotic initiation factor 4B iPGM_NPF06415.3 −263.4 BPG-independent PGAM N-terminus (iPGM_N) p450PF00067.11 −105 Cytochrome P450 zf-AN1 PF01428.6 0 AN1-like Zinc fingerzf-C3HC4 PF00097.13 16.9 Zinc finger, C3HC4 type (RING finger) zf-CCCHPF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and similar) zf-CCHCPF00098.12 17.9 Zinc knuckle zf-CSL PF05207.3 2.9 CSL zinc finger

Example 5 Plasmid Construction for Transferring Recombinant DNA

This example illustrates the construction of plasmids for transferringrecombinant DNA into the nucleus of a plant cell which can beregenerated into a transgenic crop plant of this invention. Primers forPCR amplification of protein coding nucleotides of recombinant DNA aredesigned at or near the start and stop codons of the coding sequence, inorder to eliminate most of the 5′ and 3′ untranslated regions. DNA ofinterest, i.e. each DNA identified in Table 1 and the DNA for theidentified homologous genes, are cloned and amplified by PCR prior toinsertion into the insertion site the base vector.

A. Corn Transformation Vector

Elements of an exemplary common expression vector, pMON93093 areillustrated in Table 18. The exemplary base vector which is especiallyuseful for corn transformation is illustrated in FIG. 2 and assembledusing technology known in the art. The DNA of interest are inserted in aexpression vector at the insertion site between the intron 1 of rice act1 gene and the termination sequence of PinII gene.

TABLE 18 pMON93093 Coordinates of function name annotation SEQ ID NO:19940 Agro B-AGRtu.right border Agro right border 11364-11720transforamtion sequence, essential for transfer of T-DNA. Gene ofE-Os.Act1 upstream promoter  19-775 interest region of the rice actinexpression 1 gene cassette E-CaMV.35S.2xA1-B3 duplicated35S A1-B3 788-1120 domain without TATA box P-Os.Act1 promoter region of the1125-1204 rice actin 1 gene L-Ta.Lhcb1 5′ untranslated leader 1210-1270of wheat major chlorophyll a/b binding protein I-Os.Act1 first intronand flanking 1287-1766 UTR exon sequences from the rice actin 1 geneT-St.Pis4 3′ non-translated region 1838-2780 of the potato proteinaseinhibitor II gene which functions to direct polyadenylation of the mRNAPlant P-Os.Act1 Promoter from the rice 2830-3670 selectable actin 1 genemarker L-Os.Act1 first exon of the rice 3671-3750 expression actin 1gene cassette I-Os.Act1 first intron and flanking 3751-4228 UTR exonsequences from the rice actin 1 gene TS-At.ShkG-CTP2 Transit peptideregion 4238-4465 of Arabidopsis EPSPS CR-AGRtu.aroA- Synthetic CP4coding 4466-5833 CP4.nat region with dicot preferred codon usage.T-AGRtu.nos A 3′ non-translated 5849-6101 region of the nopalinesynthase gene of Agrobacterium tumefaciens Ti plasmid which functions todirect polyadenylation of the mRNA. Agro B-AGRtu.left border Agro leftborder 6168-6609 transformation sequence, essential for transfer ofT-DNA. Maintenance OR-Ec.oriV-RK2 The vegetative origin of 6696-7092 inE. coli replication from plasmid RK2. CR-Ec.rop Coding region for8601-8792 represser of primer from the ColE1 plasmid. Expression of thisgene product interferes with primer binding at the origin ofreplication, keeping plasmid copy number low. OR-Ec.ori-ColE1 Theminimal origin of 9220-9808 replication from the E. coli plasmid ColE1.P-Ec.aadA-SPC/STR remoter for Tn7 10339-10380 adenylyltransferase(AAD(3″)) CR-Ec.aadA-SPC/STR Coding region for Tn7 10381-11169adenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycinresistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 11170-11227adenylyltransferase (AAD(3″)) gene of E. coli.

B. Soybean Transformation Vector

Plasmids for use in transformation of soybean are also prepared.Elements of an exemplary common expression vector plasmid pMON82053 areshown in Table 19 below. This exemplary soybean or canola transformationbase vector illustrated in FIG. 3 is assembled using the technologyknown in the art. Recobminant DNA of interest, i.e. each DNA identifiedin Table 1 and the DNA for the identified homologous genes, is clonedand amplified by PCR prior to insertion into the insertion site the basevector at the insertion site between the enhanced 35S CaMV promoter andthe termination sequence of cotton E6 gene.

TABLE 19 pMON82053 Coordinates of function name annotation SEQ ID NO:19941 Agro B-AGRtu.left border Agro left border sequence, 6144-6585transforamtion essential for transfer of T-DNA. Plant P-At.Act7 Promoterfrom the arabidopsis actin 6624-7861 selectable 7 gene marker L-At.Act75′UTR of Arabidopsis Act7 gene expression I-At.Act7 Intron from theArabidopsis actin7 cassette gene TS-At.ShkG-CTP2 Transit peptide regionof Arabidopsis 7864-8091 EPSPS CR-AGRtu.aroA-CP4.nno_At Synthetic CP4coding region with 8092-9459 dicot preferred codon usage. T-AGRtu.nos A3′ non-translated region of the 9466-9718 nopaline synthase gene ofAgrobacterium tumefaciens Ti plasmid which functions to directpolyadenylation of the mRNA. Gene of P-CaMV.35S-enh Promoter for 35S RNAfrom CaMV  1-613 interest containing a duplication of the −90 expressionto −350 region. cassette T-Gb.E6-3b 3′ untranslated region from thefiber  688-1002 protein E6 gene of sea-island cotton; Agro B-AGRtu.rightborder Agro right border sequence, essential 1033-1389 transformationfor transfer of T-DNA. Maintenance OR-Ec.oriV-RK2 The vegetative originof replication 5661-6057 in E. coli from plasmid RK2. CR-Ec.rop Codingregion for represser of primer 3961-4152 from the ColE1 plasmid.Expression of this gene product interferes with primer binding at theorigin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1The minimal origin of replication 2945-3533 from the E. coli plasmidColE1. P-Ec.aadA-SPC/STR romoter for Tn7 adenylyltransferase 2373-2414(AAD(3″)) CR-Ec.aadA-SPC/STR Coding region for Tn7 1584-2372adenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycinresistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 1526-1583adenylyltransferase (AAD(3″)) gene of E. coli.

C. Cotton Transformation Vector

Plasmids for use in transformation of cotton are also prepared. Elementsof an exemplary common expression vector plasmid pMON99053 are shown inTable 20 below and FIG. 4. Primers for PCR amplification of proteincoding nucleotides of recombinant DNA are designed at or near the startand stop codons of the coding sequence, in order to eliminate most ofthe 5′ and 3′ untranslated regions. Each recombinant DNA coding for aprotein identified in Table 1 is amplified by PCR prior to insertioninto the insertion site within the gene of interest expression cassetteof one of the base.

TABLE 20 Coordinates of function name annotation SEQ ID NO: 19942 AgroB-AGRtu.right border Agro right border sequence, 11364-11720transforamtion essential for transfer of T- DNA. Gene of Exp-CaMV.35S-Enhanced version of the 35S 7794-8497 interest enh + ph.DnaK RNApromoter from CaMV expression plus the petunia hsp70 5′ cassetteuntranslated region T-Ps.RbcS2-E9 The 3′ non-translated region  67-699of the pea RbcS2 gene which functions to direct polyadenylation of themRNA. Plant Exp-CaMV.35S Promoter and 5′ untranslated  730-1053selectable region of the 35S RNA from marker CaMV expressionCR-Ec.nptII-Tn5 Neomycin 1087-1881 cassette Phosphotransferase II genethat confers resistance to neomycin and kanamycin T-AGRtu.nos A 3′non-translated region of 1913-2165 the nopaline synthase gene ofAgrobacterium tumefaciens Ti plasmid which functions to directpolyadenylation of the mRNA. Agro B-AGRtu.left border Agro left bordersequence, 2211-2652 transformation essential for transfer of T- DNA.Maintenance in OR-Ec.oriV-RK2 The vegetative origin of 2739-3135 E. colireplication from plasmid RK2. CR-Ec.rop Coding region for represser4644-4835 of primer from the ColE1 plasmid. Expression of this geneproduct interferes with primer binding at the origin of replication,keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of5263-5851 replication from the E. coli plasmid ColE1. P-Ec.aadA-SPC/STRromoter for Tn7 6382-6423 adenylyltransferase (AAD(3″))CR-Ec.aadA-SPC/STR Coding region for Tn7 6424-7212 adenylyltransferase(AAD(3″)) conferring spectinomycin and streptomycin resistance.T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 7213-7270 adenylyltransferase(AAD(3″)) gene of E. coli.

Example 6 Corn Plant Transformation

This example illustrates the production and identification of transgeniccorn cells in seed of transgenic corn plants having an enhancedagronomic trait, i.e. enhanced nitrogen use efficiency, increased yield,enhanced water use efficiency, enhanced tolerance to cold and/orimproved seed compositions as compared to control plants. Transgeniccorn cells are prepared with recombinant DNA expressing each of theprotein encoding DNAs listed in Table 1 by Agrobacterium-mediatedtransformation using the corn transformation vectors pMON93093 asdisclosed in Example 6. Corn transformation is effected using methodsdisclosed in U.S. Patent Application Publication 2004/0344075 A1 wherecorn embryos are inoculated and co-cultured with the Agrobacteriumtumefaciens strain ABI and the corn transformation vector. To regeneratetransgenic corn plants the transgenic callus resulting fromtransformation is placed on media to initiate shoot development inplantlets which are transferred to potting soil for initial growth in agrowth chamber followed by a mist bench before transplanting to potswhere plants are grown to maturity. The plants are self fertilized andseed is harvested for screening as seed, seedlings or progeny R2 plantsor hybrids, e.g., for yield trials in the screens indicated above.

Many transgenic events which survive to fertile transgenic plants thatproduce seeds and progeny plants do not exhibit an enhanced agronomictrait. The transgenic plants and seeds having the transgenic cells ofthis invention which have recombinant DNA imparting the enhancedagronomic traits are identified by screening for nitrogen useefficiency, yield, water use efficiency, cold tolerance and improvedseed composition.

Example 7 Soybean Plant Transformation

This example illustrates the production and identification of transgenicsoybean cells in seed of transgenic soybean plants having an enhancedagronomic trait, i.e. enhanced nitrogen use efficiency, increased yield,enhanced water use efficiency, enhanced tolerance to cold and/orimproved seed compositions as compared to control plants. Transgenicsoybean cells are prepared with recombinant DNA expressing each of theprotein encoding DNAs listed in Table 1 by Agrobacterium-mediatedtransformation using the soybean transformation vectors pMON82053disclosed in Example 7. Soybean transformation is effected using methodsdisclosed in U.S. Pat. No. 6,384,301 where soybean meristem explants arewounded then inoculated and co-cultured with the soybean transformationvector, then transferred to selection media for 6-8 weeks to allowselection and growth of transgenic shoots.

The transformation is repeated for each of the protein encoding DNAsidentified in Table 1.

Transgenic shoots producing roots are transferred to the greenhouse andpotted in soil. Many transgenic events which survive to fertiletransgenic plants that produce seeds and progeny plants do not exhibitan enhanced agronomic trait. The transgenic plants and seeds having thetransgenic cells of this invention which have recombinant DNA impartingthe enhanced agronomic traits are identified by screening for nitrogenuse efficiency, yield, water use efficiency, cold tolerance and improvedseed composition.

Example 8 Canola Transformation

This example illustrates plant transformation useful in producing thetransgenic canola plants of this invention and the production andidentification of transgenic seed for transgenic canola having enhancedwater use efficiency, enhanced cold tolerance, increased yield, enhancednitrogen use efficiency, enhanced seed protein and enhanced seed oil.

Tissues from in vitro grown canola seedlings are prepared and inoculatedwith overnight-grown Agrobacterium cells containing plasmid DNA with thegene of interest cassette and a plant selectable marker cassette.Following co-cultivation with Agrobacterium, the infected tissues areallowed to grow on selection to promote growth of transgenic shoots,followed by growth of roots from the transgenic shoots. The selectedplantlets are then transferred to the greenhouse and potted in soil.Molecular characterization are performed to confirm the presence of thegene of interest, and its expression in transgenic plants and progenies.Progeny transgenic plants are selected from a population of transgeniccanola events under specified growing conditions and are compared withcontrol canola plants. Control canola plants are substantially the samecanola genotype but without the recombinant DNA, for example, either aparental canola plant of the same genotype that is not transformed withthe identical recombinant DNA or a negative isoline of the transformedplant

Transgenic canola plant cells are transformed with recombinant DNA fromeach of the genes identified in Table 1. Transgenic progeny plants andseed of the transformed plant cells are screened for enhanced water useefficiency, enhanced cold tolerance, increased yield, enhanced nitrogenuse efficiency, enhanced seed protein and enhanced seed oil as reportedin Example 9.

Example 9 Selection of Transgenic Plants with Enhanced AgronomicTrait(s)

This example illustrates identification of nuclei of the invention byscreening derived plants and seeds for an enhanced trait identifiedbelow.

Many transgenic events which survive to fertile transgenic plants thatproduce seeds and progeny plants will not exhibit an enhanced agronomictrait. Populations of transgenic seed and plants prepared in Examples 6and 7 are screened to identify those transgenic events providingtransgenic plant cells with a nucleus having recombinant DNA impartingan enhanced trait. Each population is screened for enhanced nitrogen useefficiency, increased yield, enhanced water use efficiency, enhancedtolerance to cold and heat, increased level of oil and protein in seedusing assays described below. Plant cell nuclei having recombinant DNAwith each of the genes identified in Table 1 and the identified homologsare identified in plants and seeds with at least one of the enhancedtraits.

A. Selection for Enhanced Nitrogen use Efficiency

Transgenic corn plants with nuclei of the invention are planted infields with three levels of nitrogen (N) fertilizer being applied, i.e.low level (0 pounds per acre N), medium level (80 pounds per acre N) andhigh level (180 pounds per acre N). Liquid 28% or 32% UAN (Urea,Ammonium Nitrogen) are used as the N source and apply by broadcast boomand incorporate with a field cultivator with rear rolling basket in thesame direction as intended crop rows. Although there is no N applied inthe low level treatment, the soil should still be disturbed in the samefashion as the treated area. Transgenic plants and control plants can begrouped by genotype and construct with controls arranged randomly withingenotype blocks. For improved statistical analysis each type oftransgenic plant can be tested by 3 replications and across 4 locations.Nitrogen levels in the fields are analyzed before planting by collectingsample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples areanalyzed for nitrate-nitrogen, phosphorus (P), potassium (K), organicmatter and pH to provide baseline values. P, K and micronutrients areapplied based upon soil test recommendations.

Transgenic corn plants prepared in Example 6 and which exhibit a 2 to 5%yield increase as compared to control plants when grown in the highnitrogen field are selected as having nuclei of the invention.Transgenic corn plants which have at least the same or higher yield ascompared to control plants when grown in the medium nitrogen field areselected as having nuclei of the invention. Transgenic corn plantshaving a nucleus with DNA identified in Table 3 as imparting nitrogenuse efficiency (LN) and homologous DNA are selected from a nitrogen useefficiency screen as having a nucleus of this invention.

B. Selection for Increased Yield

Many transgenic plants of this invention exhibit improved yield ascompared to a control plant. Improved yield can result from enhancedseed sink potential, i.e. the number and size of endosperm cells orkernels and/or enhanced sink strength, i.e. the rate of starchbiosynthesis. Sink potential can be established very early during kerneldevelopment, as endosperm cell number and size are determined within thefirst few days after pollination.

Much of the increase in corn yield of the past several decades hasresulted from an increase in planting density. During that period, cornyield has been increasing at a rate of 2.1 bushels/acre/year, but theplanting density has increased at a rate of 250 plants/acre/year. Acharacteristic of modern hybrid corn is the ability of these varietiesto be planted at high density. Many studies have shown that a higherthan current planting density should result in more biomass production,but current germplasm does not perform well at these higher densities.One approach to increasing yield is to increase harvest index (HI), theproportion of biomass that is allocated to the kernel compared to totalbiomass, in high density plantings.

Effective yield selection of enhanced yielding transgenic corn eventsuses hybrid progeny of the transgenic event over multiple locations withplants grown under optimal production management practices, and maximumpest control. A useful target for improved yield is a 5% to 10% increasein yield as compared to yield produced by plants grown from seed for acontrol plant. Selection methods may be applied in multiple and diversegeographic locations, for example up to 16 or more locations, over oneor more planting seasons, for example at least two planting seasons tostatistically distinguish yield improvement from natural environmentaleffects. It is to plant multiple transgenic plants, positive andnegative control plants, and pollinator plants in standard plots, forexample 2 row plots, 20 feet long by 5 feet wide with 30 inches distancebetween rows and a 3 foot alley between ranges. Transgenic events can begrouped by recombinant DNA constructs with groups randomly placed in thefield. A pollinator plot of a high quality corn line is planted forevery two plots to allow open pollination when using male steriletransgenic events. A useful planting density is about 30,000plants/acre. High planting density is greater than 30,000 plants/acre,preferably about 40,000 plants/acre, more preferably about 42,000plants/acre, most preferably about 45,000 plants/acre. Each of thetransgenic corn plants and soybean plants with a nucleus of theinvention prepared in Examples 6 and 7 are screened for yieldenhancement. At least one event from each of the corn and soybean plantsis selected as having at least between 3 and 5% increase in yield ascompared to a control plant as having a nucleus of this invention.

C. Selection for Enhanced Water use Efficiency (WUE)

The following is a high-throughput method for screening for water useefficiency in a greenhouse to identify the transgenic corn plants with anucleus of this invention. This selection process imposes 3drought/re-water cycles on plants over a total period of 15 days afteran initial stress free growth period of 11 days. Each cycle consists of5 days, with no water being applied for the first four days and a waterquenching on the 5th day of the cycle. The primary phenotypes analyzedby the selection method are the changes in plant growth rate asdetermined by height and biomass during a vegetative drought treatment.The hydration status of the shoot tissues following the drought is alsomeasured. The plant height are measured at three time points. The firstis taken just prior to the onset drought when the plant is 11 days old,which is the shoot initial height (SIH). The plant height is alsomeasured halfway throughout the drought/re-water regimen, on day 18after planting, to give rise to the shoot mid-drought height (SMH). Uponthe completion of the final drought cycle on day 26 after planting, theshoot portion of the plant is harvested and measured for a final height,which is the shoot wilt height (SWH) and also measured for shoot wiltedbiomass (SWM). The shoot is placed in water at 40 degree Celsius in thedark. Three days later, the shoot is weighted to give rise to the shootturgid weight (STM). After drying in an oven for four days, the shootsare weighted for shoot dry biomass (SDM). The shoot average height (SAH)is the mean plant height across the 3 height measurements. The proceduredescribed above may be adjusted for +/− ˜ one day for each step giventhe situation.

To correct for slight differences between plants, a size correctedgrowth value is derived from SIH and SWH. This is the Relative GrowthRate (RGR). Relative Growth Rate (RGR) is calculated for each shootusing the formula [RGR %=(SWH−SIH)/((SWH+SIH)/2)×100]. Relative watercontent (RWC) is a measurement of how much (%) of the plant was water atharvest. Water Content (RWC) is calculated for each shoot using theformula [RWC %=(SWM−SDM)/(STM−SDM)×100]. Fully watered corn plants ofthis age run around 98% RWC.

Transgenic corn plants and soybean plants prepared in Examples 6 and 7are screened for water use efficiency. Transgenic plants having at leasta 1% increase in RGR and RWC as compared to control plants areidentified as having enhanced water used efficiency and are selected ashaving a nucleus of this invention. Transgenic corn and soybean plantshaving in their nucleus DNA identified in Table 3 as imparting droughttolerance improvement (DS) and homologous DNA are identified as showingincreased water use efficiency as compared to control plants and areselected as having a nucleus of this invention.

D. Selection for Growth Under Cold Stress

Cold germination assay—Three sets of seeds are used for the assay. Thefirst set consists of positive transgenic events (F1 hybrid) where thegenes of the present invention are expressed in the seed. The secondseed set is nontransgenic, wild-type negative control made from the samegenotype as the transgenic events. The third set consisted of two coldtolerant and one cold sensitive commercial check lines of corn. Allseeds are treated with a fungicide “Captan” (MAESTRO® 80DF Fungicide,Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan isapplied per 45 g of corn seeds by mixing it well and drying thefungicide prior to the experiment.

Corn kernels are placed embryo side down on blotter paper within anindividual cell (8.9×8.9 cm) of a germination tray (54×36 cm). Ten seedsfrom an event are placed into one cell of the germination tray. Eachtray can hold 21 transgenic events and 3 replicates of wildtype(LH244SDms+LH59), which is randomized in a complete block design. Forevery event there are five replications (five trays). The trays areplaced at 9.7 C for 24 days (no light) in a Convrion® growth chamber(Conviron Model PGV36, Controlled Environments, Winnipeg, Canada). Twohundred and fifty milliliters of deionized water are added to eachgermination tray. Germination counts are taken 10th, 11th, 12th, 13th,14th, 17th, 19th, 21st, and 24th day after start date of the experiment.Seeds are considered germinated if the emerged radicle size is 1 cm.From the germination counts germination index is calculated.

The germination index is calculated as per:

Germination index=(Σ([T+1−n _(i) ]*[P _(i) −P _(i-1)]))/T

where T is the total number of days for which the germination assay isperformed. The number of days after planting is defined by n. “i”indicated the number of times the germination had been counted,including the current day. P is the percentage of seeds germinatedduring any given rating. Statistical differences are calculated betweentransgenic events and wild type control. After statistical analysis, theevents that show a statistical significance at the p level of less than0.1 relative to wild-type controls will advance to a secondary coldselection. The secondary cold screen is conducted in the same manner ofthe primary selection only increasing the number of repetitions to ten.Statistical analysis of the data from the secondary selection isconducted to identify the events that show a statistical significance atthe p level of less than 0.05 relative to wild-type controls.

Transgenic corn plants and soybean plants prepared in Examples 6 and 7are screened for water use efficiency. Transgenic plants having at leasta 5% increase in germination index as compared to control plants areidentified as having enhanced cold stress tolerance and are selected ashaving a nucleus of this invention. Transgenic corn and soybean plantshaving in their nucleus DNA identified in Table 3 as imparting coldtolerance improvement (CK or CS) and homologous DNA are identified asshowing increased cold stress tolerance as compared to control plantsand are selected as having a nucleus of this invention.

E. Screens for Transgenic Plant Seeds with Increased Protein and/or OilLevels

The following is a high-throughput selection method for identifyingplant seeds with improvement in seed composition using the Infratec®1200 series Grain Analyzer, which is a near-infrared transmittancespectrometer used to determine the composition of a bulk seed sample.Near infrared analysis is a non-destructive, high-throughput method thatcan analyze multiple traits in a single sample scan. An NIR calibrationfor the analytes of interest is used to predict the values of an unknownsample. The NIR spectrum is obtained for the sample and compared to thecalibration using a complex chemometric software package that provides apredicted values as well as information on how well the sample fits inthe calibration.

Infratec® Model 1221, 1225, or 1227 analyzer with transport module byFoss North America is used with cuvette, item #1000-4033, Foss NorthAmerica or for small samples with small cell cuvette, Foss standardcuvette modified by Leon Girard Co. Corn and soy check samples ofvarying composition maintained in check cell cuvettes are supplied byLeon Girard Co. NIT collection software is provided by MaximumConsulting Inc. Calculations are performed automatically by thesoftware. Seed samples are received in packets or containers withbarcode labels from the customer. The seed is poured into the cuvettesand analyzed as received.

TABLE 21 Typical sample(s): Whole grain corn and soybean seedsAnalytical time to run method: Less than 0.75 min per sample Totalelapsed time per run: 1.5 minute per sample Typical and minimum sampleCorn typical: 50 cc; minimum 30 cc size: Soybean typical: 50 cc; minimum5 cc Typical analytical range: Determined in part by the specificcalibration. Corn - moisture 5-15%, oil 5-20%, protein 5-30%, starch50-75%, and density 1.0-1.3%. Soybean - moisture 5-15%, oil 15-25%, andprotein 35-50%.

Transgenic corn plants and soybean plants prepared in Examples 6 and 7are screened for increased protein and oil in seed. Transgenic inbredcorn and soybean plants having an increase of at least 1 percentagepoint in the total percent seed protein or at least 0.3 percentage pointin total seed oil and transgenic hybrid corn plants having an increaseof at least 0.4 percentage point in the total percent seed protein ascompared to control plants are identified as having enhanced seedprotein or enhanced seed oil and are selected as having a nucleus ofthis invention.

Example 10 Cotton Transgenic Plants with Enhanced Agronomic Traits

Cotton transformation is performed as generally described in WO0036911and in U.S. Pat. No. 5,846,797. Transgenic cotton plants containing eachof the recombinant DNA having a sequence of SEQ ID NO: 1 through SEQ IDNO: 197 are obtained by transforming with recombinant DNA from each ofthe genes identified in Table 1. Progeny transgenic plants are selectedfrom a population of transgenic cotton events under specified growingconditions and are compared with control cotton plants. Control cottonplants are substantially the same cotton genotype but without therecombinant DNA, for example, either a parental cotton plant of the samegenotype that was not transformed with the identical recombinant DNA ora negative isoline of the transformed plant. Additionally, a commercialcotton cultivar adapted to the geographical region and cultivationconditions, i.e. cotton variety ST474, cotton variety FM 958, and cottonvariety Siokra L-23, are used to compare the relative performance of thetransgenic cotton plants containing the recombinant DNA. The specifiedculture conditions are growing a first set of transgenic and controlplants under “wet” conditions, i.e. irrigated in the range of 85 to 100percent of evapotranspiration to provide leaf water potential of −14 to−18 bars, and growing a second set of transgenic and control plantsunder “dry” conditions, i.e. irrigated in the range of 40 to 60 percentof evapotranspiration to provide a leaf water potential of −21 to −25bars. Pest control, such as weed and insect control is applied equallyto both wet and dry treatments as needed. Data gathered during the trialincludes weather records throughout the growing season includingdetailed records of rainfall; soil characterization information; anyherbicide or insecticide applications; any gross agronomic differencesobserved such as leaf morphology, branching habit, leaf color, time toflowering, and fruiting pattern; plant height at various points duringthe trial; stand density; node and fruit number including node abovewhite flower and node above crack boll measurements; and visual wiltscoring. Cotton boll samples are taken and analyzed for lint fractionand fiber quality. The cotton is harvested at the normal harvesttimeframe for the trial area. Enhanced water use efficiency is indicatedby increased yield, improved relative water content, enhanced leaf waterpotential, increased biomass, enhanced leaf extension rates, andimproved fiber parameters.

The transgenic cotton plants of this invention are identified from amongthe transgenic cotton plants by agronomic trait screening as havingincreased yield and enhanced water use efficiency.

Example 11 Monocot and Dicot Plant Transformation for the Suppression ofEndogeneous Protein

This example illustrates monocot and dicot plant transformation toproduce nuclei of this invention in cells of a transgenic plant bytransformation where the recombinant DNA suppresses the expression of anendogenous protein identified by Pfam, SNF5, LMBR1, TFIIS_M, TFIIS_C, orGlyco_transf_8. Corn callus and soybean tissue are transformed asdescribe in Examples 6 and 7 using recombinant DNA in the nucleus withDNA that transcribes to RNA that forms double-stranded RNA targeted toan endogenous gene with DNA encoding the protein. The genes for whichthe double-stranded RNAs are targeted are the native gene in corn andsoybean that are homolog of the genes encoding the protein with an aminoacid sequence of SEQ ID NO:200, 201, 205, 207, 211, and 394.

Populations of transgenic corn plants and soybean plants prepared inExamples 6 and 7 with DNA for suppressing a gene identified in Table 3as providing an enhanced trait by gene suppression are screened toidentify an event from those plants with a nucleus of the invention byselecting the trait identified in this specification.

1. A plant cell nucleus with stably integrated, recombinant DNAcomprising a promoter that is functional in plant cells and that isoperably linked to DNA from a plant, bacteria or yeast that encodes aprotein having at least one domain of amino acids in a sequence thatexceeds the Pfam gathering cutoff for amino acid sequence alignment witha protein domain family identified by a Pfam name selected from thegroup of Pfam names consisting of L51_S25_CI-B8, iPGM_N, WD40,BPL_LipA_LipB, DUF676, AAA, S_locus_glycop, ArfGap, Rotamase,Metallophos, CMAS, Sugar_tr, LMBR1, RrnaAD, NAF, BolA, Pkinase, C2,FA_hydroxylase, p450, Complex1_30kDa, Histone, DUF822, PEP-utilizers,PCI, ETC_C1_NDUFA5, 2-Hacid_dh, Tryp_alpha_amyl, PK_C, MAP65_ASE1,FBPase, SWIB, Ank, Ribosomal_S8e, 2-Hacid_dh_C, SMC_N, GTP_cyclohydro2,PfkB, ORMDL, ADH_zinc_N, SWIM, TrkA_N, HLH, GH3, SNF5, Ceramidase_alk,Ribonuclease T2, Complex1_49kDa, Gp_dh_C, Aldo_ket_red, zf-AN1, TFIIS_C,MFS_1, Thioredoxin, DUF1005, LEA_3, Sterol_MT_C, Gp_dh_N, TFIIS_M,PAN_2, BPL_C, DUF26, Aa_trans, ACT, ADH_N, NAD_binding_1,Auxin_inducible, B_lectin, Anti-silence, Response_reg, 14-3-3, LRRNT_2,GDC-P, zf-CCHC, NPH3, TPR_1, TFIIA, DHBP_synthase, UQ_con, TPR_2, TPT,F-box, adh_short, Cyclin_C, Na_H_Exchanger, AA_permease, MtN3_slv, TIM,NDK, Pantoate_transf, Allene_ox_cyc, Cyclin_N, Methyltransf_11, CBM_20,Methyltransf_12, Rhodanese, Glycolytic, Actin, Usp, eIF-4B,Glyco_transf_8, BURP, Alpha-amylase, F420_oxidored, EGF_CA, Kelch_1,PGAM, Aminotran_1_2, Kelch_2, UPF0261, CoA_binding, DUF868,Peptidase_S10, Lung_7-TM_R, Oleosin, Sad1_UNC, Gln-synt_C, LSM,NTP_transferase, Metalloenzyme, Prenylcys_lyase, Subtilisin_N, SAM_1,DUF298, ESCRT-III, DNA_pol_E_B, Aminotran_3, NAD_Gly3P_dh_N, Gln-synt_N,MMR_HSR1. DUF588, zf-CCCH, DnaJ, Pkinase_Tyr, Cupin_2, LRR_1, Cupin_3,zf-CSL, FAR1, HD, FH12, APC8, PTR2, MannoseP_isomer, Rib_5-P_isom_A,DUF1336, Phosphorylase, DUFF1191, Asp, Mit_rib_S27, PAP_fibrillin,DUF1195, Aldedh, zf-C3HC4, PPR, PK, PurA, RMMBL, HTH_11, Tim17, and PBDwherein said Pfam gathering cutoff for said protein domain families arestated in Table 17; wherein said plant cell nucleus is selected byscreening a population of transgenic plants that have said recombinantDNA and express said protein for an enhanced trait as compared tocontrol plants that do not have said recombinant DNA in their nuclei;and wherein said enhanced trait is selected from group of enhancedtraits consisting of enhanced water use efficiency, enhanced coldtolerance, enhanced heat tolerance, enhanced resistance to saltexposure, enhanced shade tolerance, increased yield, enhanced nitrogenuse efficiency, enhanced seed protein and enhanced seed oil.
 2. Theplant cell nucleus of claim 1 wherein said protein has an amino acidsequence with at least 90% identity to a consensus amino acid sequencein the group of consensus amino acid sequences consisting of theconsensus amino acid sequence constructed for SEQ ID NO: 198 andhomologs thereof listed in Table 2 through the consensus amino acidsequence constructed for SEQ ID NO: 394 and homologs thereof listed inTable
 2. 3. The plant cell nucleus of claim 1 wherein said protein isselected from the group of proteins identified in Table
 1. 4. A plantcell nucleus with stably integrated, recombinant DNA to suppress thelevel of an endogenous protein having at least one domain of amino acidsin a sequence that exceeds the Pfam gathering cutoff for amino acidsequence alignment with a protein domain family identified by Pfam namein the group of Pfam names consisting of SNF5, LMBR1, TFIIS_M, TFIIS_C,and Glyco_transf_8, wherein the Pfam gathering cutoff for said proteindomain families is stated in Table 17 wherein said plant cell nucleus isselected by screening a population of transgenic plants with saidrecombinant DNA and have the level of said endogenous protein suppressedfor an enhanced trait as compared to control plants that do not havesaid recombinant DNA; and wherein said enhanced trait is selected fromthe group of enhanced traits consisting of enhanced water useefficiency, enhanced cold tolerance, enhanced heat tolerance, enhancedresistance to salt exposure, enhanced shade tolerance, increased yield,enhanced nitrogen use efficiency, enhanced seed protein and enhancedseed oil.
 5. The plant cell nucleus of claim 1 further comprising DNAexpressing a protein that provides tolerance from exposure to anherbicide applied at levels that are lethal to a wild type of said plantcell.
 6. The plant cell nucleus of claim 5 wherein the agent of saidherbicide is a glyphosate, dicamba, or glufosinate compound.
 7. Atransgenic plant cell or plant comprising a plurality of plant cellswith a plant cell nucleus of claim
 1. 8. The transgenic plant cell orplant of claim 7 which is homozygous for said recombinant DNA.
 9. Atransgenic seed comprising a plurality of plant cells with a plant cellnucleus of claim
 1. 10. The transgenic seed of claim 9 from a corn,soybean, cotton, canola, alfalfa, wheat or rice plant.
 11. Thetransgenic corn seed of claim 10 wherein said seed can produce cornplants that are resistant to disease from the Mal de Rio Cuarto virus orthe Puccinia sorghi fungus or both.
 12. A transgenic pollen graincomprising a haploid derivative of a plant cell nucleus of claim
 1. 13.A method for manufacturing non-natural, transgenic seed that can be usedto produce a crop of transgenic plants with an enhanced trait resultingfrom expression of stably-integrated recombinant DNA in a nucleus ofclaim 1, wherein said method for manufacturing said transgenic seedcomprising: (a) screening a population of plants for said enhanced traitand said recombinant DNA, wherein individual plants in said populationcan exhibit said trait at a level less than, essentially the same as orgreater than the level that said trait is exhibited in control plantswhich do not express the recombinant DNA, (b) selecting from saidpopulation one or more plants that exhibit said trait at a level greaterthan the level that said trait is exhibited in control plants, (c)verifying that said recombinant DNA is stably integrated in saidselected plants, (d) analyzing tissue of said selected plant todetermine the production or suppression of a protein having the functionof a protein encoded by nucleotides having a sequence selected from thegroup consisting of one of SEQ ID NO:198-394; and (e) collecting seedfrom said selected plant.
 14. The method of claim 13 wherein plants insaid population further comprise DNA expressing a protein that providestolerance to exposure to an herbicide applied at levels that are lethalto wild type plant cells, and wherein said selecting is effected bytreating said population with said herbicide.
 15. The method of claim 14wherein said herbicide comprises a glyphosate, dicamba, or glufosinatecompound.
 16. The method of claim 15 wherein said selecting is effectedby identifying plants with said enhanced trait.
 17. The method of claim16 wherein said seed is corn, soybean, cotton, alfalfa, wheat or riceseed.
 18. A method of producing hybrid corn seed comprising: (a)acquiring hybrid corn seed from a herbicide tolerant corn plant whichalso has stably-integrated, recombinant DNA in a nucleus of claim 1; (b)producing corn plants from said hybrid corn seed, wherein a fraction ofthe plants produced from said hybrid corn seed is homozygous for saidrecombinant DNA, a fraction of the plants produced from said hybrid cornseed is hemizygous for said recombinant DNA, and a fraction of theplants produced from said hybrid corn seed has none of said recombinantDNA; (c) selecting corn plants which are homozygous and hemizygous forsaid recombinant DNA by treating with an herbicide; (d) collecting seedfrom herbicide-treated-surviving corn plants and planting said seed toproduce further progeny corn plants; (e) repeating steps (c) and (d) atleast once to produce an inbred corn line; (f) crossing said inbred cornline with a second corn line to produce hybrid seed.